Combination of dr5 agonist and anti-pd-1 antagonist and methods of use

ABSTRACT

Provided are methods and compositions for treating cancer using an effective amount of a PD-1 antagonist (e.g., an antibody) in combination with a DR4 or DR5 agonist (e.g., an antibody).

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/993,687, filed on May 31, 2018 (now allowed), which is a continuationof U.S. patent application Ser. No. 14/774,382, filed on Sep. 10, 2015,(now abandoned), which is a U.S. National Stage Entry of InternationalApplication No. PCT/US2014/024208, filed Mar. 12, 2014, and which claimsthe benefit of U.S. Provisional Application No. 61/783,184, filed onMar. 14, 2013. The entire contents of these applications areincorporated herein by reference in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jul. 27, 2021, isnamed “MXI_529USCN2_Sequence_Listing.txt” and is 24,915 bytes in size.The Sequence Listing is being submitted by EFS Web and is herebyincorporated by reference into the specification.

BACKGROUND

The National Cancer Institute has estimated that in the United Statesalone, 1 in 3 people will be struck with cancer during their lifetime.Moreover, approximately 50% to 60% of people contracting cancer willeventually succumb to the disease. The widespread occurrence of thisdisease underscores the need for improved anticancer regimens for thetreatment of malignancy.

Programmed Cell Death 1 (PD-1) is a cell surface signaling receptor thatplays a critical role in the regulation of T cell activation andtolerance (Keir M E, et al., Annu Rev Immunol 2008; 26:677-704). It is atype I transmembrane protein and together with BTLA, CTLA-4, ICOS andCD28, comprise the CD28 family of T cell co-stimulatory receptors. PD-1is primarily expressed on activated T cells, B cells, and myeloid cells(Dong H, et al., Nat Med 1999; 5:1365-1369). It is also expressed onnatural killer (NK) cells (Terme M, et al., Cancer Res 2011;71:5393-5399). PD-1 is highly expressed on tumor infiltratinglymphocytes, and its ligands are up-regulated on the cell surface ofmany different tumors (Dong H, et al., Nat Med 2002; 8:793-800).Multiple murine cancer models have demonstrated that binding of ligandto PD-1 results in immune evasion. In addition, blockade of thisinteraction results in anti-tumor activity.

Two cell surface glycoprotein ligands for PD-1 have been identified,PD-L1 and PD-L2, and have been shown to downregulate T cell activationand cytokine secretion upon binding to PD-1 (Freeman et al. (2000) J ExpMed 192:1027-34; Latchman et al. (2001) Nat Immunol 2:261-8; Carter etal. (2002) Eur J Immunol 32:634-43; Ohigashi et al. (2005) Clin CancerRes 11:2947-53). Both PD-L1 (B7-H1) and PD-L2 (B7-DC) are B7 homologsthat bind to PD-1, but do not bind to other CD28 family members (Blanket al. (2004). Expression of PD-L1 on the cell surface has also beenshown to be upregulated through IFN-gamma stimulation.

PD-L1 expression has been found in several murine and human cancers,including human lung, ovarian and colon carcinoma and various myelomas(Iwai et al. (2002) PNAS 99:12293-7; Ohigashi et al. (2005) Clin CancerRes 11:2947-53). PD-L1 has been suggested to play a role in tumorimmunity by increasing apoptosis of antigen-specific T-cell clones (Donget al. (2002) Nat Med 8:793-800). It has also been suggested that PD-L1might be involved in intestinal mucosal inflammation and inhibition ofPD-L1 suppresses wasting disease associated with colitis (Kanai et al.(2003) J Immunol 171:4156-63).

TRAIL (tumor necrosis factor (TNF)-related apoptosis-inducing ligand) isa member of the TNF superfamily with the ability to induce apoptosis oftumor cells. At least five receptors for TRAIL have been identified. DR4(TRAIL-R1) and DR5 (TRAIL-R2) are apoptosis-inducing receptors, whicheach contain an intracellular death domain (see e.g., Pan G, et al.,Science. 1997; 276:111-113, Pan G, et al., Science. 1997; 277:815-818,Sheridan J P, et al., Science. 1997; 277:818-821, and Walczak H, et al.,EMBO J. 1997; 16:5386-5397). Upon receptor activation, DR4 and DR5recruit FAS associated protein with death domain (FADD) and caspase-8 toform the death-inducing signaling complex (DISC), which activatescaspase-8, subsequently leading to the activation of executionercaspases such as caspase-3 that induce apoptosis (see, e.g., Kischkel FC, et al., Immunity. 2000; 12:611-620, Thomas L R, et al., J Biol Chem.2004; 279:32780-32785, Thomas L R, et al., J Biol Chem. 2004;279:52479-52486, Varfolomeev E, et al., J Biol Chem. 2005;280:40599-40608, Ashkenazi A., Nat Rev Cancer. 2002; 2:420-430, andThorburn A. Cell Signal. 2004; 16:139-144).

TRAIL and agonistic antibodies that recognize TRAIL receptorspreferentially kill tumor cells and induce potent anti-tumor activity ina variety of experimental models (see, Griffith T S, et al., Curr OpinImmunol. 1998; 10:559-563, Ashkenazi A, et al., J Clin Invest. 1999;104:155-162, Walczak H, et al, Nat Med. 1999; 5:157-163, Chuntharapai A,et al., J Immunol. 2001; 166:4891-4898, and Ichikawa K, et al., Nat Med.2001; 7:954-960). Administration of TRAIL to mice bearing human tumorsactively suppressed tumor progression and improved survival of theanimal (Walczak H, et al, Nat Med. 1999; 5:157-163). Accordingly,agonists against DR4 or DR5 by activating apoptosis are becomingdramatically meaningful as candidates for the treatment of cancer.

SUMMARY OF THE INVENTION

The present inventors have discovered for the first time thatco-administration of a DR5 agonist (e.g., an antibody) and an anti-PD-1antagonist (e.g., an antibody) effectively inhibits tumor growth invivo, even synergistically. Accordingly, it is an object of the presentinvention to provide improved methods for treating subjects with cancer.Specifically, it is an object of the invention to provide efficaciouscombination treatment regimens wherein a DR5 agonist is combined with ananti-PD-1 antagonist for the treatment of cancer.

The present invention provides a method for the treatment of cancer in asubject by co-administering an effective amount of a PD-1 antagonist andan agent that induces apoptosis in cancer cells, e.g., an agent thatengages the DR4 or DR5 receptor, such as a DR4 or DR5 agonist.

Suitable anti-DR5 agonists for use in the methods of the invention,include, without limitation, ligands, antibodies (e.g., monoclonalantibodies and bispecific antibodies) and multivalent agents. In oneembodiment, the DR5 agonist is an antibody selected from the groupconsisting of Lexatumumab (also known as ETR2-ST01), Tigatuzumab (alsoknown as CS-1008), Conatumumab (also known as AMG 655), Drozitumab,HGSTR2J/KMTRS, and LBY-135. In another embodiment, the DR5 agonist is amultivalent agent (e.g., TAS266). In a further embodiment, the DR5agonist is a ligand (e.g., a TNF-related apoptosis-inducing ligand(TRAIL), such as a recombinant human TRAIL, e.g., Dulanermin (also knownas AMG951)).

Suitable PD-1 antagonists for use in the methods of the invention,include, without limitation, ligands, antibodies (e.g., monoclonalantibodies and bispecific antibodies), and multivalent agents. In oneembodiment, the PD-1 antagonist is a fusion protein, e.g., an Fc fusionprotein, such as AMP-244. In one embodiment, the PD-1 antagonist is ananti-PD-1 or anti-PD-L1 antibody. In another embodiment, the PD-1antagonist is an antibody, such as MK-3475 or CT-011.

An exemplary anti-PD-1 antibody is 5C4 (referred to as 5C4 in WO2006/121168; also known as MDX-1106, ONO-4538, and Nivolumab) comprisingheavy and light chains having the sequences shown in SEQ ID NOs: 11 and12, respectively, or antigen binding fragments and variants thereof. Inother embodiments, the antibody comprises the heavy and light chain CDRsor VRs of 5C4. Accordingly, in one embodiment, the antibody comprisesthe CDR1, CDR2, and CDR3 domains of the VH region of 5C4 having thesequence shown in SEQ ID NO: 13, and the CDR1, CDR2 and CDR3 domains ofthe VL region of 5C4 having the sequence shown in SEQ ID NO:15. Inanother embodiment, the antibody comprises the heavy chain CDR1, CDR2and CDR3 domains having the sequences set forth in SEQ ID NOs: 17, 18,and 19, respectively, and the light chain CDR1, CDR2 and CDR3 domainshaving the sequences set forth in SEQ ID NOs: 20, 21, and 22,respectively. In another embodiment, the antibody comprises VH and/or VLregions having the amino acid sequences set forth in SEQ ID NO: 13and/or SEQ ID NO: 15, respectively. In another embodiment, the antibodycomprises the heavy chain variable (VH) and/or light chain variable (VL)regions encoded by the nucleic acid sequences set forth in SEQ ID NO: 14and/or SEQ ID NO: 16, respectively.

In another embodiment, the antibody competes for binding with, and/orbinds to the same epitope on PD-1 as, the above-mentioned antibodies. Inanother embodiment, the antibody has at least about 90% variable regionamino acid sequence identity with the above-mentioned antibodies (e.g.,at least about 90%, 95% or 99% variable region identity with SEQ ID NO:13 or SEQ ID NO: 15).

In one embodiment, the PD-1 antagonist is an anti-PD-L1 antibody, suchas MEDI4736 (also known as Anti-B7-H1) or MPDL3280A (also known asRG7446). An exemplary anti-PD-L1 antibody is 12A4 (referred to as 12A4in WO 2007/005874 and U.S. Pat. No. 7,943,743). In one embodiment, theantibody comprises the heavy and light chain CDRs or VRs of 12A4.Accordingly, in one embodiment, the antibody comprises the CDR1, CDR2,and CDR3 domains of the VH region of 12A4 having the sequence shown inSEQ ID NO: 1, and the CDR1, CDR2 and CDR3 domains of the VL region of5C4 having the sequence shown in SEQ ID NO: 3. In another embodiment,the antibody comprises the heavy chain CDR1, CDR2 and CDR3 domainshaving the sequences set forth in SEQ ID NOs: 5, 6, and 7, respectively,and the light chain CDR1, CDR2 and CDR3 domains having the sequences setforth in SEQ ID NOs: 8, 9, and 10, respectively. In another embodiment,the antibody comprises VH and/or VL regions having the amino acidsequences set forth in SEQ ID NO: 1 and/or SEQ ID NO: 3, respectively.In another embodiment, the antibody comprises the heavy chain variable(VH) and/or light chain variable (VL) regions encoded by the nucleicacid sequences set forth in SEQ ID NO: 2 and/or SEQ ID NO: 4,respectively. In another embodiment, the antibody competes for bindingwith, and/or binds to the same epitope on PD-L1 as, the above-mentionedantibodies. In another embodiment, the antibody has at least about 90%variable region amino acid sequence identity with the above-mentionedantibodies (e.g., at least about 90%, 95% or 99% variable regionidentity with SEQ ID NO: 1 or SEQ ID NO: 3).

In one embodiment, the invention provides a method of treating cancer ina subject, the method comprising administering to the subject aneffective amount of a PD-1 antagonist and a DR5 agonist, wherein

(a) the PD-1 antagonist is an anti-PD-1 antibody comprising the CDR1,CDR2 and CDR3 domains in a heavy chain variable region having thesequence set forth in SEQ ID NO: 13, and the CDR1, CDR2 and CDR3 domainsin a light chain variable region having the sequence set forth in SEQ IDNO: 15; and(b) the DR5 agonist is an antibody.

In another embodiment, the invention provides a method cancer in asubject, the method comprising administering to the subject an effectiveamount of a PD-1 antagonist and a DR5 agonist, wherein

(a) the PD-1 antagonist is an anti-PD-L1 antibody comprising the CDR1,CDR2 and CDR3 domains in a heavy chain variable region having thesequence set forth in SEQ ID NO: 1, and the CDR1, CDR2 and CDR3 domainsin a light chain variable region having the sequence set forth in SEQ IDNO: 3; and(b) the DR5 agonist is an antibody.

The efficacy of the treatment methods provided herein can be assessedusing any suitable means. In one embodiment, the treatment produces atleast one therapeutic effect selected from the group consisting ofreduction in size of a tumor, reduction in number of metastasic lesionsover time, complete response, partial response, and stable disease. Inanother embodiment, administration of a PD-1 antagonist and a DR5agonist results in at least a 1, 1.25, 1.50, 1.75, 2, 2.25, 2.50, 2.75,3, 3.25, 3.5, 3.75, or 4-fold reduction in tumor volume, e.g., relativeto treatment with the PD-1 antagonist or DR5 agonist alone, or relativeto tumor volume before initiation of treatment. In another embodiment,administration of the PD-1 antagonist and DR5 agonist results in atleast a 1-fold, 2-fold, or more preferably a 3-fold reduction in tumorvolume, e.g., relative to treatment with the PD-1 antagonist or DR5agonist alone, or relative to tumor volume before initiation oftreatment. In a further embodiment, administration of a PD-1 antagonistand a DR5 agonist results in tumor growth inhibition of at least 50%,60%, 70% or 80%, e.g., relative to treatment with the PD-1 antagonist orDR5 agonist alone, or relative to tumor volume before initiation oftreatment. In certain embodiments, tumor volume is reduced by 50%, 60%,70%, 80%, 90% or more, e.g., relative to tumor size before initiation ofthe treatment.

The PD-1 antagonist and DR5 agonist can be administered accordingly to asuitable dosage, route (e.g., intravenous, intraperitoneal,intramuscular, intrathecal or subcutaneous). The antagonist and agonistcan also be administered according to any suitable schedule. Forexample, the antagonist and agonist can be simultaneously administeredin a single formulation. Alternatively, the antagonist and agonist canbe formulated for separate administration, wherein they are administeredconcurrently or sequentially. In one embodiment, the PD-1 antagonist isadministered prior to administration of the DR5 agonist. In anotherembodiment, the DR5 agonist is administered prior to administration ofthe PD-1 antagonist. In a further embodiment, the DR5 agonist and thePD-1 antagonist are administered simultaneously.

In one embodiment, the cancer is a cancer selected from the groupconsisting of leukemia, lymphoma, blastoma, carcinoma and sarcoma. Inanother embodiment, the cancer is selected from the group consisting ofchronic myeloid leukemia, acute lymphoblastic leukemia, Philadelphiachromosome positive acute lymphoblastic leukemia (Ph+ ALL), squamouscell carcinoma, small-cell lung cancer, non-small cell lung cancer,glioma, gastrointestinal cancer, renal cancer, ovarian cancer, livercancer, colorectal cancer, endometrial cancer, kidney cancer, prostatecancer, thyroid cancer, neuroblastoma, pancreatic cancer, glioblastomamultiforme, cervical cancer, stomach cancer, bladder cancer, hepatoma,breast cancer, colon carcinoma, and head and neck cancer, gastriccancer, germ cell tumor, pediatric sarcoma, sinonasal natural killer,multiple myeloma, acute myelogenous leukemia (AML), and chroniclymphocytic leukemia (CML).

Additional agents and therapies can be administered in combination withthe agonists and antagonists described herein. In one embodiment, themethods comprise administration of an additional therapeutic agent(e.g., a cyotoxin or chemotherapeutic agent.

Also provided herein are compositions comprising a PD-1 antagonist and aDR5 agonist. In one embodiment, the antagonist and/or agonist is aligand, antibody (e.g., monoclonal antibody or bispecific antibody) ormultivalent agent. In another embodiment, the PD-1 antagonist is ananti-PD-1 antibody comprising the heavy and light chain CDRs or VRs of5C4. In another embodiment, the PD-1 antagonist is an anti-PD-L1antibody comprising the heavy and light chain CDRs or VRs of 12A4.

Further provided are kits for treating a cancer in a subject, the kitcomprising:

(a) a dose of a PD-1 antagonist;

(b) a dose of a DR5 agonist; and

(c) instructions for using the PD-1 antagonist and DR5 agonist in themethods described herein. In one embodiment, the DR5 agonist is anantibody. In another embodiment, the PD-1 antagonist is an antibody. Inparticular embodiment, the PD-1 antagonist is an anti-PD-1 antibodycomprising the CDR1, CDR2 and CDR3 domains in a heavy chain variableregion having the sequence set forth in SEQ ID NO: 13, and the CDR1,CDR2 and CDR3 domains in a light chain variable region having thesequence set forth in SEQ ID NO: 15. In another particular embodiment,the PD-1 antagonist is an anti-PD-L1 antibody comprising antibodycomprises the CDR1, CDR2 and CDR3 domains in a heavy chain variableregion having the sequence set forth in SEQ ID NO: 1, and the CDR1, CDR2and CDR3 domains in a light chain variable region having the sequenceset forth in SEQ ID NO: 3.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph depicting the median tumor volume in mice (mm³) afteradministration of a control, an anti-DR5 antibody, an anti-PD-1antibody, or combination of both an anti-DR5 antibody, an anti-PD-1antibody, up to 23 days post implant.

FIGS. 2A-2H depict the tumor volume in individual mice administered acontrol (FIG. 2A), an anti-PD-1 antibody on day 6 post-implant (FIG.2B), an anti-PD-1 antibody on day 8 post-implant (FIG. 2C), an anti-PD-1antibody on day 9 post-implant (FIG. 2D), an anti-DR5 antibody on day 8post-implant (FIG. 2E), an anti-DR5 antibody on day 8 post-implant incombination with an anti-PD-1 antibody on day 6 post-implant (FIG. 2F),an anti-DR5 antibody on day 8 post-implant in combination with ananti-PD-1 antibody on day 8 post-implant (FIG. 2G), and an anti-DR5antibody on day 8 post-implant in combination with an anti-PD-1 antibodyon day 9 post-implant (FIG. 2H).

FIG. 3 is a graph depicting the percent body weight change followingadministration of a control, an anti-DR5 monoclonal antibody, ananti-PD-1 antibody, or combination of both an anti-DR5 monoclonalantibody and anti-PD-1 antibody, up to 24 days post implant.

DETAILED DESCRIPTION OF THE INVENTION

As described herein, the invention is based on the discovery thatco-administration of a DR5 agonist (e.g., an antibody) and a PD-1antagonist (e.g., an antibody) effectively inhibits tumor growth invivo, even synergistically. Accordingly, the present invention providesa method for the treatment of cancer in a subject which comprisesadministering to a subject (e.g., human) an effective amount of a PD-1antagonist and a DR5 agonist.

I. Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by the skilled artisan.Although any methods and compositions similar or equivalent to thosedescribed herein can be used in practice or testing of the presentinvention, the preferred methods and compositions are described herein.

As used herein, the term “subject” or “patient” is a human patient(e.g., a patient having cancer).

A “solid tumor” includes, for example, sarcoma, melanoma, carcinoma,prostate carcinoma, lung carcinoma, colon carcinoma, or other solidtumor cancer.

The terms “cancer”, “cancerous”, or “malignant” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth. Examples of cancer include, for example,leukemia, lymphoma, blastoma, carcinoma and sarcoma. More particularexamples of such cancers include chronic myeloid leukemia, acutelymphoblastic leukemia, Philadelphia chromosome positive acutelymphoblastic leukemia (Ph+ ALL), squamous cell carcinoma, small-celllung cancer, non-small cell lung cancer, glioma, gastrointestinalcancer, renal cancer, ovarian cancer, liver cancer, colorectal cancer,endometrial cancer, kidney cancer, prostate cancer, thyroid cancer,neuroblastoma, pancreatic cancer, glioblastoma multiforme, cervicalcancer, stomach cancer, bladder cancer, hepatoma, breast cancer, coloncarcinoma, and head and neck cancer, gastric cancer, germ cell tumor,pediatric sarcoma, sinonasal natural killer, multiple myeloma, acutemyelogenous leukemia (AML), and chronic lymphocytic leukemia (CML).

As used herein, “effective treatment” refers to treatment producing abeneficial effect, e.g., amelioration of at least one symptom of adisease or disorder. A beneficial effect can take the form of animprovement over baseline, i.e., an improvement over a measurement orobservation made prior to initiation of therapy according to the method.A beneficial effect can also take the form of arresting, slowing,retarding, or stabilizing of a deleterious progression of a marker ofcancer. Effective treatment may refer to alleviation of at least onesymptom of cancer. Such effective treatment may, e.g., reduce patientpain, reduce the size and/or number of lesions, may reduce or preventmetastasis of a tumor, and/or may slow tumor growth.

The term “effective amount” refers to an amount of an agent thatprovides the desired biological, therapeutic, and/or prophylacticresult. That result can be reduction, amelioration, palliation,lessening, delaying, and/or alleviation of one or more of the signs,symptoms, or causes of a disease, or any other desired alteration of abiological system. In reference to solid tumors, an effective amountcomprises an amount sufficient to cause a tumor to shrink and/or todecrease the growth rate of the tumor (such as to suppress tumor growth)or to prevent or delay other unwanted cell proliferation. In someembodiments, an effective amount is an amount sufficient to delay tumordevelopment. In some embodiments, an effective amount is an amountsufficient to prevent or delay tumor recurrence. An effective amount canbe administered in one or more administrations. The effective amount ofthe drug or composition may: (i) reduce the number of cancer cells; (ii)reduce tumor size; (iii) inhibit, retard, slow to some extent and maystop cancer cell infiltration into peripheral organs; (iv) inhibit(i.e., slow to some extent and may stop tumor metastasis; (v) inhibittumor growth; (vi) prevent or delay occurrence and/or recurrence oftumor; and/or (vii) relieve to some extent one or more of the symptomsassociated with the cancer. In one example, an “effective amount” is theamount of a PD-1 antagonist (e.g., an antibody) and DR5 agonist antibody(e.g., an antibody), in combination, to effect a significant decrease incancer or slowing of progression of cancer, such as an advanced solidtumor.

As used herein, the term “antagonist” refers to a molecule which blocks(e.g., reduces or prevents) a biological activity.

As used herein, the term “agonist” refers to a molecule that triggers(e.g., initiates or promotes), partially or fully enhances, stimulatesor activates one or more biological activities. Agonists often mimic theaction of a naturally occurring substance. Whereas an agonist causes anaction, an antagonist blocks the action of the agonist.

As used herein, the term “ligand” refers to a molecule that forms acomplex with a biomolecule (e.g., a receptor) to serve a biologicalpurpose. In a narrower sense, is a signal triggering molecule, bindingto a site on a target protein. The binding occurs by intermolecularforces, such as ionic bonds, hydrogen bonds and van der Waals forces.The docking (association) is usually reversible (dissociation). Actualirreversible covalent binding between a ligand and its target moleculeis rare in biological systems. Ligand binding to a receptor (receptorprotein) alters its chemical conformation (three dimensional shape). Theconformational state of a receptor protein determines its functionalstate.

As used herein, the terms “synergy”, “therapeutic synergy”, and“synergistic effect” refer to a phenomenon where treatment of patientswith a combination of therapeutic agents (e.g., PD-1 antagonist incombination with DR5 agonist) manifests a therapeutically superioroutcome to the outcome achieved by each individual constituent of thecombination used at its optimum dose (see, e.g., T. H. Corbett et al.,1982, Cancer Treatment Reports, 66, 1187). In this context atherapeutically superior outcome is one in which the patients either a)exhibit fewer incidences of adverse events while receiving a therapeuticbenefit that is equal to or greater than that where individualconstituents of the combination are each administered as monotherapy atthe same dose as in the combination, or b) do not exhibit dose-limitingtoxicities while receiving a therapeutic benefit that is greater thanthat of treatment with each individual constituent of the combinationwhen each constituent is administered in at the same doses in thecombination(s) as is administered as individual components. In xenograftmodels, a combination, used at its maximum tolerated dose, in which eachof the constituents will be present at a dose generally not exceedingits individual maximum tolerated dose, manifests therapeutic synergywhen decrease in tumor growth achieved by administration of thecombination is greater than the value of the decrease in tumor growth ofthe best constituent when the constituent is administered alone.

As used herein, the term “antibody” includes whole antibodies and anyantigen binding fragment (i.e., “antigen-binding fragments” (also knownas “antigen-binding portions”)) or single chains thereof. Wholeantibodies are glycoproteins comprising at least two heavy (H) chainsand two light (L) chains inter-connected by disulfide bonds. Each heavychain is comprised of a heavy chain variable region (abbreviated hereinas VH) and a heavy chain constant region. The heavy chain constantregion is comprised of three domains, C_(H)1, C_(H)2 and C_(H)3. Eachlight chain is comprised of a light chain variable region (abbreviatedherein as V_(L)) and a light chain constant region. The light chainconstant region is comprised of one domain, C_(L). The V_(H) and V_(L)regions can be further subdivided into regions of hypervariability,termed complementarity determining regions (CDR), interspersed withregions that are more conserved, termed framework regions (FR). EachV_(H) and V_(L) is composed of three CDRs and four FRs, arranged fromamino-terminus to carboxy-terminus in the following order: FR1, CDR1,FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and lightchains contain a binding domain that interacts with an antigen. Theconstant regions of the antibodies can mediate the binding of theimmunoglobulin to host tissues or factors, including various cells ofthe immune system (e.g., effector cells) and the first component (C1q)of the classical complement system. The term “antibody” also encompasseschimeric antibodies, humanized antibodies, fully human antibodies, aswell as multimeric forms of antibodies, such as minibodies, bis-scFv,diabodies, triabodies, tetrabodies and chemically conjugated Fab′multimers.

The term “antibody fragment” (also referred to as “antigen-bindingfragment” or “antigen-binding portion”), as used herein, refers to oneor more fragments of an antibody that retain the ability to specificallybind to an antigen. It has been shown that the antigen-binding functionof an antibody can be performed by fragments of a full-length antibody.Examples of binding fragments encompassed within the term“antigen-binding fragment” of an antibody include (i) a Fab fragment, amonovalent fragment consisting of the V_(L), V_(H), C_(L) and C_(H)1domains; (ii) a F(ab′)₂ fragment, a bivalent fragment is essentially aFab with part of the hinge region (see, FUNDAMENTAL IMMUNOLOGY (Pauled., 3.sup.rd ed. 1993); (iv) a Fd fragment consisting of the V_(H) andC_(H)1 domains; (v) a Fv fragment consisting of the V_(L) and V_(H)domains of a single arm of an antibody, (vi) a dAb fragment (Ward etal., (1989) Nature 341:544-546), which consists of a V_(H) domain; (vii)an isolated complementarity determining region (CDR); and (viii) ananobody (also known as a single-domain antibody (sdAb)), which is aheavy chain variable region containing a single variable domain and twoconstant domains.

Single domain antibodies include V_(H)H fragments (single-domainantibodies engineered from heavy-chain antibodies found in camelids, aswell as VNAR fragments (single-domain antibodies obtained fromheavy-chain antibodies (IgNAR, ‘immunoglobulin new antigen receptor’) ofcartilaginous fishes).

“Antigen binding scaffolds” are proteins that bind specifically to atarget (or antigen) or epitope, such as proteins comprising an Ig foldor an Ig-like fold, e.g., the DR5 binding proteins described inWO2009/058379 and WO2011/130328, Antibodies or antigen binding fragmentsthereof are also antigen binding scaffolds. Antigen binding scaffoldscan be monovalent, multivalent, e.g., bivalent, trivalent, tetravalent,or bind 5, 6 or more epitopes. Multivalent antigen binding scaffolds canbe monospecific or multispecific, i.e., binding to multiple (at least 2,3, 4 or 5) epitopes that are different from one another. For example, amultivalent monospecific antigen binding scaffold is a protein thatbinds to at least 2, 3, 4 or 5 identical epitopes, and may be a proteincomprising at least 2, 3, 4 or 5 identical antigen binding portions. Forexample, DR5 binding scaffolds may comprise 2-10, e.g., 2-6, 2-5, 2-4 or2-3 DR5 binding portions, which may be the same or different from oneanother.

A multivalent antibody includes antibodies comprising at least 2, 3, 4,5, 6, 7, 8, 9, 10 or more antigen binding portions of antibodies, whichantigen binding portions may comprise a portion of a heavy chain and aportion of a light chain. An antigen binding portion may be on a singlepolypeptide or comprise more than one polypeptide. For example, amultivalent antibody may comprise from 2-10 antigen binding portions,which may be the same or different from each other. A multivalentantibody may be monospecific or multispecific. A multispecific antibodymay be bispecific, trispecific, tetraspecific or bind to 5 or moredifferent epitopes.

Furthermore, although the two domains of the Fv fragment, V_(L) andV_(H), are coded for by separate genes, they can be joined, usingrecombinant methods, by a synthetic linker that enables them to be madeas a single protein chain in which the V_(L) and V_(H) regions pair toform monovalent molecules (known as single chain Fv (scFv); see e.g.,Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc.Natl. Acad. Sci. USA 85:5879-5883). Such single chain antibodies arealso intended to be encompassed within the term “antigen-bindingfragment” of an antibody. These antibody fragments are obtained usingconventional techniques known to those with skill in the art, and thefragments are screened for utility in the same manner as are intactantibodies.

As used herein, an antigen binding scaffold that “specifically binds” toan antigen or epitope thereof is an antigen binding scaffold that bindsto the antigen or epitope thereof with a K_(D) of 10⁻⁷ M, 5×10⁻⁸ M, 10⁻⁸M, 5×10⁻⁹ M, 10⁻⁹ M, 5×10⁻¹⁰ M, 10⁻¹⁰ M, 5×10⁻¹¹ M, 10⁻¹¹ M, 5×10⁻¹² M,10⁻¹² M or less. For example, an antigen binding scaffold thatspecifically binds to DR5 is an antigen binding scaffold that binds toDR5 with a K_(D) of 10⁻⁷ M, 5×10⁻⁸ M, 10⁻⁸ M, 5×10⁻⁹ M, 10⁻⁹ M, 5×10⁻¹⁰M, 10⁻¹⁰ M, 5×10⁻¹¹ M, 10⁻¹¹ M, 5×10⁻¹² M, 10⁻¹² M or less. For example,an antibody that “specifically binds to human PD-1” or “specificallybinds to human PD-L1” is intended to refer to an antibody that binds tohuman PD-1 or PD-L1, respectively, with a K_(D) of 10⁻⁷ M, 5×10⁻⁸ M,10⁻⁸ M, 5×10⁻⁹ M, 10⁻⁹ M, 5×10⁻¹⁰ M, 10⁻¹⁰ M, 5×10⁻¹¹ M, 10⁻¹¹ M,5×10⁻¹² M, 10⁻¹² M or less. An antigen binding scaffold that comprises 2or more regions binding to an antigen or epitope may bind specificallyto the antigen or epitope even it has a lower affinity of binding to theantigen or epitope than the ranges provided above, as it will bind tothe antigen or epitope with increased avidity.

A “bispecific” or “bifunctional antibody” is an artificial hybridantibody having two different heavy/light chain pairs and two differentbinding sites. Bispecific antibodies can be produced by a variety ofmethods including fusion of hybridomas or linking of Fab′ fragments.See, e.g., Songsivilai & Lachmann, Clin. Exp. Immunol. 79:315-321(1990); Kostelny et al., J. Immunol. 148, 1547-1553 (1992).

The term “monoclonal antibody” or “monoclonal antibody composition,” asused herein, refers to an antibody or a composition of antibodies thatdisplays a single binding specificity and affinity for a particularepitope. Accordingly, the term “human monoclonal antibody” or“monoclonal antibody composition” refers to an antibody or a compositionof antibodies which displays a single binding specificity and which hasvariable and optional constant regions derived from human germlineimmunoglobulin sequences. In one embodiment, human monoclonal antibodiesare produced by a hybridoma which includes a B cell obtained from atransgenic non-human animal, e.g., a transgenic mouse, having a genomecomprising a human heavy chain transgene and a light chain transgenefused to an immortalized cell.

The term “epitope” or “antigenic determinant” refers to a site on anantigen to which an immunoglobulin or antibody specifically binds.Epitopes can be formed both from contiguous amino acids or noncontiguousamino acids juxtaposed by tertiary folding of a protein. Epitopes formedfrom contiguous amino acids are typically retained on exposure todenaturing solvents, whereas epitopes formed by tertiary folding aretypically lost on treatment with denaturing solvents. An epitopetypically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or15 amino acids in a unique spatial conformation. Methods of determiningspatial conformation of epitopes include techniques in the art and thosedescribed herein, for example, x-ray crystallography and 2-dimensionalnuclear magnetic resonance (see, e.g., Epitope Mapping Protocols inMethods in Molecular Biology, Vol. 66, G. E. Morris, Ed. (1996)).

The term “epitope mapping” refers to the process of identification ofthe molecular determinants for antibody-antigen recognition.

The term “binds to the same epitope,” with reference to two or moreantibodies, means that the antibodies compete for binding to an antigenand bind to the same, overlapping, or encompassing continuous ordiscontinuous segments of amino acids. Those of skill in the artunderstand that the phrase “binds to the same epitope” does notnecessarily mean that the antibodies bind to exactly the same aminoacids. The precise amino acids to which the antibodies bind can differ.For example, a first antibody can bind to a segment of amino acids thatis completely encompassed by the segment of amino acids bound by asecond antibody. In another example, a first antibody binds one or moresegments of amino acids that significantly overlap the one or moresegments bound by the second antibody. For the purposes herein, suchantibodies are considered to “bind to the same epitope.”

Accordingly, also, encompassed by the present invention are antibodiesthat bind to an epitope which comprises all or a portion of an epitoperecognized by the particular antibodies described herein (e.g., the sameor an overlapping region or a region between or spanning the region).

Also encompassed by the present invention are antibodies that bind thesame epitope and/or antibodies that compete for binding with theantibodies described herein.

Antibodies that recognize the same epitope or compete for binding can beidentified using routine techniques. Such techniques include, forexample, an immunoassay, which shows the ability of one antibody toblock the binding of another antibody to a target antigen, i.e., acompetitive binding assay. Competitive binding is determined in an assayin which the immunoglobulin under test inhibits specific binding of areference antibody to a common antigen. Numerous types of competitivebinding assays are known, for example: solid phase direct or indirectradioimmunoassay (RIA), solid phase direct or indirect enzymeimmunoassay (EIA), sandwich competition assay (see Stahli et al.,Methods in Enzymology 9:242 (1983)); solid phase direct biotin-avidinEIA (see Kirkland et al., J. Immunol. 137:3614 (1986)); solid phasedirect labeled assay, solid phase direct labeled sandwich assay (seeHarlow and Lane, Antibodies: A Laboratory Manual, Cold Spring HarborPress (1988)); solid phase direct label RIA using I-125 label (see Morelet al., Mol. Immunol. 25(1):7 (1988)); solid phase direct biotin-avidinEIA (Cheung et al., Virology 176:546 (1990)); and direct labeled RIA.(Moldenhauer et al., Scand. J. Immunol. 32:77 (1990)). Typically, suchan assay involves the use of purified antigen bound to a solid surfaceor cells bearing either of these, an unlabeled test immunoglobulin and alabeled reference immunoglobulin. Competitive inhibition is measured bydetermining the amount of label bound to the solid surface or cells inthe presence of the test immunoglobulin. Usually the test immunoglobulinis present in excess. Usually, when a competing antibody is present inexcess, it will inhibit specific binding of a reference antibody to acommon antigen by at least 50-55%, 55-60%, 60-65%, 65-70% 70-75% ormore.

Other techniques include, for example, epitope mapping methods, such as,x-ray analyses of crystals of antigen:antibody complexes which providesatomic resolution of the epitope. Other methods monitor the binding ofthe antibody to antigen fragments or mutated variations of the antigenwhere loss of binding due to a modification of an amino acid residuewithin the antigen sequence is often considered an indication of anepitope component. In addition, computational combinatorial methods forepitope mapping can also be used. These methods rely on the ability ofthe antibody of interest to affinity isolate specific short peptidesfrom combinatorial phage display peptide libraries. The peptides arethen regarded as leads for the definition of the epitope correspondingto the antibody used to screen the peptide library. For epitope mapping,computational algorithms have also been developed which have been shownto map conformational discontinuous epitopes.

Chimeric molecules (or fusion molecules) comprising an antigen bindingdomain, or equivalent, fused to another polypeptide or molecule are alsoencompassed by the present invention. For example, the polypeptides maybe fused or conjugated to an antibody Fc region, or portion thereof(e.g., an Fc fusion protein). The antibody portion fused to apolypeptide may comprise the constant region, hinge region, CH1 domain,CH2 domain, and CH3 domain or any combination of whole domains orportions thereof. The polypeptides may also be fused or conjugated tothe above antibody portions to form multimers. For example, Fc portionsfused to the polypeptides of the present invention can form dimersthrough disulfide bonding between the Fc portions. Higher multimericforms can be made by fusing the polypeptides to portions of IgA and IgM.Methods for fusing or conjugating the polypeptides of the presentinvention to antibody portions are known in the art. See, e.g., U.S.Pat. Nos. 5,336,603, 5,622,929, 5,359,046, 5,349,053, 5,447,851, and5,112,946; EP 307,434; EP 367,166; PCT Publication Nos. WO 96/04388 andWO 91/06570; Ashkenazi et al., Proc. Natl. Acad. Sci. USA,88:10535-10539 (1991); Zheng et al., J. Immunol., 154:5590-5600 (1995);and Vil et al., Proc. Natl. Acad. Sci. USA, 89:11337-11341 (1992).

As used herein, the term “immunoconjugate” refers to an antibody linkedto a therapeutic moiety, such as a cytotoxin, a drug or a radioisotope.When conjugated to a cytotoxin, these antibody conjugates are referredto as “immunotoxins.” A cytotoxin or cytotoxic agent includes any agentthat is detrimental to (e.g., kills) cells. Examples include taxol,cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin,etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin,daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin,actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine,tetracaine, lidocaine, propranolol, and puromycin and analogs orhomologs thereof. Therapeutic agents include, but are not limited to,antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine,cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g.,mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) andlomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol,streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP)cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine and vinblastine). Antibodies use in the presentinvention can be conjugated to a radioisotope, e.g., radioactive iodine,to generate cytotoxic radiopharmaceuticals for treating cancer.

Immunoconjugates can be used to modify a given biological response, andthe drug moiety is not to be construed as limited to classical chemicaltherapeutic agents. For example, the drug moiety may be a protein orpolypeptide possessing a desired biological activity. Such proteins mayinclude, for example, an enzymatically active toxin, or active fragmentthereof, such as abrin, ricin A, Pseudomonas exotoxin, or diphtheriatoxin; a protein such as tumor necrosis factor or interferon-γ; or,biological response modifiers such as, for example, lymphokines,interleukin-1 (“IL-1”), interleukin-2 (“IL-2”), interleukin-6 (“IL-6”),granulocyte macrophage colony stimulating factor (“GM-CSF”), granulocytecolony stimulating factor (“G-CSF”), or other growth factors.

Techniques for conjugating such therapeutic moiety to antibodies arewell known, see, e.g., Arnon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss,Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53(Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84:Biological And Clinical Applications, Pinchera et al. (eds.), pp.475-506 (1985); “Analysis, Results, And Future Prospective Of TheTherapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., “ThePreparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”,Immunol. Rev., 62:119-58 (1982).

As used herein, the term “multivalent” refers to a recombinant moleculethat incorporates more than two biologically active segments. Theprotein fragments forming the multivalent molecule optionally may belinked through a polypeptide linker which attaches the constituent partsand permits each to function independently.

“About” as used herein when referring to a measurable value such as anamount, a temporal duration, and the like, is meant to encompassvariations of .+−.20% or .+−.10%, more preferably .+−.5%, even morepreferably .+−.1%, and still more preferably .+−.0.1% from the specifiedvalue, as such variations are appropriate to perform the disclosedmethods.

“Percent (%) amino acid sequence identity” herein is defined as thepercentage of amino acid residues in a candidate sequence that areidentical with the amino acid residues in a selected sequence, afteraligning the sequences and introducing gaps, if necessary, to achievethe maximum percent sequence identity, and not considering anyconservative substitutions as part of the sequence identity. Alignmentfor purposes of determining percent amino acid sequence identity can beachieved in various ways that are within the skill in the art, forinstance, using publicly available computer software such as BLAST,BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Those skilled inthe art can determine appropriate parameters for measuring alignment,including any algorithms needed to achieve maximal alignment over thefull-length of the sequences being compared.

For purposes herein, the % amino acid sequence identity of a given aminoacid sequence A to, with, or against a given amino acid sequence B(which can alternatively be phrased as a given amino acid sequence Athat has or comprises a certain % amino acid sequence identity to, with,or against a given amino acid sequence B) is calculated as follows: 100times the fraction X/Y where X is the number of amino acid residuesscored as identical matches by a sequence alignment program, such asBLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR), in that program'salignment of A and B, and where Y is the total number of amino acidresidues in B. It will be appreciated that where the length of aminoacid sequence A is not equal to the length of amino acid sequence B, the% amino acid sequence identity of A to B will not equal the % amino acidsequence identity of B to A.

II. PD-1 Antagonists

As used herein, the terms “Programmed Death 1,” “Programmed Cell Death1,” “Protein PD-1,” “PD-1,” PD1,” “PDCD1,” “hPD-1” and “hPD-I” are usedinterchangeably, and include variants, isoforms, species homologs ofhuman PD-1, and analogs having at least one common epitope with humanPD-1. The complete human PD-1 sequence can be found under GenBankAccession No. U64863 (SEQ ID NO:23).

As used herein, the terms “Programmed Cell Death 1 Ligand 1”, “PD-L1”,“PDL1”, “PDCD1L1”, “PDCD1LG1”, “CD274”, “B7 homolog 1”, “B7-H1”, “B7-H”,and “B7H1” are used interchangeably, and include variants, isoforms,species homologs of human PDL-1, and analogs having at least one commonepitope with human PDL-1. The complete human PD-L1 amino acidsequence—isoform a precursor—can be found under GenBank Accession No.NP_054862.1 (SEQ ID NO:24). The complete human PD-L1 amino acidsequence—isoform b precursor—can be found under GenBank Accession No.NP_001254635.1 (SEQ ID NO:25).

The protein Programmed Death 1 (PD-1) is an inhibitory member of theCD28 family of receptors, that also includes CD28, CTLA-4, ICOS andBTLA. PD-1 is expressed on activated B cells, T cells, and myeloid cells(Agata et al., supra; Okazaki et al. (2002) Curr. Opin. Immunol. 14:391779-82; Bennett et al. (2003) J Immunol 170:711-8). The initialmembers of the family, CD28 and ICOS, were discovered by functionaleffects on augmenting T cell proliferation following the addition ofmonoclonal antibodies (Hutloff et al. (1999) Nature 397:263-266; Hansenet al. (1980) Immunogenics 10:247-260). PD-1 was discovered throughscreening for differential expression in apoptotic cells (Ishida et al.(1992) EMBO J 11:3887-95). The other members of the family, CTLA-4 andBTLA, were discovered through screening for differential expression incytotoxic T lymphocytes and TH1 cells, respectively. CD28, ICOS andCTLA-4 all have an unpaired cysteine residue allowing forhomodimerization. In contrast, PD-1 is suggested to exist as a monomer,lacking the unpaired cysteine residue characteristic in other CD28family members.

The PD-1 gene is a 55 kDa type I transmembrane protein that is part ofthe Ig gene superfamily (Agata et al. (1996) Int Immunol 8:765-72). PD-1contains a membrane proximal immunoreceptor tyrosine inhibitory motif(ITIM) and a membrane distal tyrosine-based switch motif (ITSM) (Thomas,M. L. (1995) J Exp Med 181:1953-6; Vivier, E and Daeron, M (1997)Immunol Today 18:286-91). Although structurally similar to CTLA-4, PD-1lacks the MYPPPY motif (SEQ ID NO: 27) that is critical for B7-1 andB7-2 binding.

Consistent with PD-1 being an inhibitory member of the CD28 family, PD-1deficient animals develop various autoimmune phenotypes, includingautoimmune cardiomyopathy and a lupus-like syndrome with arthritis andnephritis (Nishimura et al. (1999) Immunity 11:141-51; Nishimura et al.(2001) Science 291:319-22). Additionally, PD-1 has been found to play arole in autoimmune encephalomyelitis, systemic lupus erythematosus,graft-versus-host disease (GVHD), type I diabetes, and rheumatoidarthritis (Salama et al. (2003) J Exp Med 198:71-78; Prokunina andAlarcon-Riquelme (2004) Hum Mol Genet 13:R143; Nielsen et al. (2004)Lupus 13:510). In a murine B cell tumor line, the ITSM of PD-1 was shownto be essential to block BCR-mediated Ca²⁺-flux and tyrosinephosphorylation of downstream effector molecules (Okazaki et al. (2001)PNAS 98:13866-71).

Two ligands for PD-1 have been identified, PD-L1 and PD-L2, that havebeen shown to downregulate T cell activation upon binding to PD-1(Freeman et al. (2000) J Exp Med 192:1027-34; Latchman et al. (2001) NatImmunol 2:261-8; Carter et al. (2002) Eur J Immunol 32:634-43). BothPD-L1 and PD-L2 are B7 homologs that bind to PD-1, but do not bind toother CD28 family members. PD-L1 is abundant in a variety of humancancers (Dong et al. (2002) Nat. Med. 8:787-9). The interaction betweenPD-1 and PD-L1 results in a decrease in tumor infiltrating lymphocytes,a decrease in T-cell receptor mediated proliferation, and immune evasionby the cancerous cells (Dong et al. (2003) J. Mol. Med. 8:281-7; Blanket al. (2005) Cancer Immunol. Immunother. 54:307-314; Konishi et al.(2004) Clin. Cancer Res. 10:5094-100). Immune suppression can bereversed by inhibiting the local interaction of PD-1 with PD-L1, and theeffect is additive when the interaction of PD-1 with PD-L2 is blocked aswell (Iwai et al. (2002) Proc. Nat'l. Acad. Sci. USA 99:12293-7; Brownet al. (2003) J. Immunol. 170:1257-66).

The methods of the present invention involve the use of a PD-1antagonist (e.g., an antibody in combination with a DR5 agonist (e.g.,an antibody), for treating cancer. Accordingly, PD-1 antagonists of theinvention bind to ligands of PD-1 and interfere with, reduce, or inhibitthe binding of one or more ligands to the PD-1 receptor, or binddirectly to the PD-1 receptor, without engaging in signal transductionthrough the PD-1 receptor. In one embodiment, the PD-1 antagonist bindsdirectly to PD-1 and blocks PD-1 inhibitory signal transduction. Inanother embodiment the PD-1 antagonist binds to one or more ligands ofPD-1 (e.g., PD-L1 and PD-L2) and reduces or inhibits the ligand(s) fromtriggering inhibitory signal transduction through the PD-1. In oneembodiment, the PD-1 antagonist binds directly to PD-L1, inhibiting orpreventing PD-L1 from binding to PD-1, thereby blocking PD-1 inhibitorysignal transduction.

PD-1 antagonists used in the methods and compositions of the presentinvention include PD-1 binding scaffold proteins and include, but arenot limited to, PD-1 ligands, antibodies and multivalent agents. In aparticular embodiment, the antagonist is a fusion protein, such asAMP-224. In another embodiment, the antagonist is an anti-PD-1 antibody(“PD-1 antibody”). Anti-human-PD-1 antibodies (or VH and/or VL domainsderived therefrom) suitable for use in the invention can be generatedusing methods well known in the art. Alternatively, art recognizedanti-PD-1 antibodies can be used. For example, antibodies MK-3475 orCT-011 can be used. Additionally, monoclonal antibodies 5C4, 17D8, 2D3,4H1, 4A11, 7D3, and 5F4, described in WO 2006/121168, the teachings ofwhich are hereby incorporated by reference, can be used. Antibodies thatcompete with any of these art-recognized antibodies for binding to PD-1also can be used.

An exemplary anti-PD-1 antibody is 5C4 comprising heavy and light chainshaving the sequences shown in SEQ ID NOs: 11 and 12, respectively, orantigen binding fragments and variants thereof. In other embodiments,the antibody comprises the heavy and light chain CDRs or variableregions of 5C4. Accordingly, in one embodiment, the antibody comprisesthe CDR1, CDR2, and CDR3 domains of the VH of 5C4 having the sequenceset forth in SEQ ID NO: 13, and the CDR1, CDR2 and CDR3 domains of theVL of 5C4 having the sequences set forth in SEQ ID NO: 15. In anotherembodiment, the antibody comprises CDR1, CDR2 and CDR3 domains havingthe sequences set forth in SEQ ID NOs: 17, 18, and 19, respectively, andCDR1, CDR2 and CDR3 domains having the sequences set forth in SEQ IDNOs: 20, 21, and 22, respectively. In another embodiment, the antibodycomprises VH and/or VL regions having the amino acid sequences set forthin SEQ ID NO: 13 and/or SEQ ID NO: 15, respectively. In anotherembodiment, the antibody comprises the heavy chain variable (VH) and/orlight chain variable (VL) regions encoded by the nucleic acid sequencesset forth in SEQ ID NO: 14 and/or SEQ ID NO: 16, respectively. Inanother embodiment, the antibody competes for binding with and/or bindsto the same epitope on PD-1 as the above-mentioned antibodies. Inanother embodiment, the antibody has at least about 90% variable regionamino acid sequence identity with the above-mentioned antibodies (e.g.,at least about 90%, 95% or 99% variable region identity with SEQ ID NO:13 or SEQ ID NO: 15).

In certain embodiments, the PD1 antibodies exhibit one or more desirablefunctional properties, such as high affinity binding to PD-1, e.g.,binding to human PD-1 with a K_(D) of 10⁻⁷ M or less; lack ofsignificant cross-reactivity to other CD28 family members, e.g., CD28,CTLA-4 and ICOS; the ability to stimulate T cell proliferation in amixed lymphocyte reaction (MLR) assay; the ability to increase IFN-7and/or IL-2 secretion in an MLR; the ability to inhibit binding of oneor more PD-1 ligands (e.g., PD-L1 and/or PD-L2) to PD-1; the ability tostimulate antigen-specific memory responses; the ability to stimulateantibody responses and/or the ability to inhibit growth of tumor cellsin vivo.

In another embodiment, the PD-1 antagonist is an anti-PD-L1 antibody.Anti-human-PD-L1 antibodies (or VH and/or VL domains derived therefrom)suitable for use in the invention can be generated using methods wellknown in the art. Alternatively, art recognized anti-PD-L1 antibodiescan be used. For example, MEDI4736 (also known as Anti-B7-H1) orMPDL3280A (also known as RG7446) can be used. Additionally, monoclonalantibodies 12A4, 3G10, 10A5, 5F8, 10H10, 1B12, 7H1, 11E6, 12B7, and 13G4described in WO 2007/005874 and U.S. Pat. No. 7,943,743, the teachingsof which are hereby incorporated by reference, can be used. Antibodiesthat compete with any of these art-recognized antibodies for binding toPD-L1 also can be used.

An exemplary anti-PD-L1 antibody is 12A4 (WO 2007/005874 and U.S. Pat.No. 7,943,743). In one embodiment, the antibody comprises the heavy andlight chain CDRs or VRs of 12A4. Accordingly, in one embodiment, theantibody comprises the CDR1, CDR2, and CDR3 domains of the VH region of12A4 having the sequence shown in SEQ ID NO: 1 and the CDR1, CDR2 andCDR3 domains of the VL region of 12A4 having the sequence shown in SEQID NO: 3. In another embodiment, the antibody comprises the heavy chainCDR1, CDR2 and CDR3 domains having the sequences set forth in SEQ IDNOs: 5, 6, and 7, respectively, and the light chain CDR1, CDR2 and CDR3domains having the sequences set forth in SEQ ID NOs: 8, 9, and 10,respectively. In another embodiment, the antibody comprises VH and/or VLregions having the amino acid sequences set forth in SEQ ID NO: 1 and/orSEQ ID NO: 3, respectively. In another embodiment, the antibodycomprises the heavy chain variable (VH) and/or light chain variable (VL)regions encoded by the nucleic acid sequences set forth in SEQ ID NO: 2and/or SEQ ID NO: 4, respectively. In another embodiment, the antibodycompetes for binding with, and/or binds to the same epitope on PD-L1 as,the above-mentioned antibodies. In another embodiment, the antibody hasat least about 90% variable region amino acid sequence identity with theabove-mentioned antibodies (e.g., at least about 90%, 95% or 99%variable region identity with SEQ ID NO: 1 or SEQ ID NO: 3).

Anti-PD-1 or anti-PD-L1 antibodies may bind to PD-1 or PD-L1,respectively, with a K_(D) of 10⁻⁷ M, 5×10⁻⁸ M, 10⁻⁸ M, 5×10⁻⁹ M, 10⁻⁹M, 5×10⁻¹⁰ M, 10⁻¹⁰ M or less.

III. DR5 Agonists

Provided herein are methods for treating cancer comprising administeringto a subject in need thereof (e.g., a subject having cancer), atherapeutically effective amount of an agent that induces apoptosis in acancer cell and a PD-1 antagonist. Exemplary apoptosis inducing agentsinclude DR proteins, such as DR4 and DR5.

As used herein, the terms “DR5” and “death receptor 5”, also known as“tumor necrosis factor receptor superfamily member 10b”, “TNFRSF10B”,“CD262”, “KILLER”, “TRICK2”, “TRICKB”, “ZTNFR9”, “TRAILR”, “TRAILR2”,“Apo-2” “TRICK2A”, “TRICK2B”, “TRAIL-R2”, “KILLER”, “KILLER/DR5”, “TR6”,“Tango-63”, “hAPO8”, and TRICK2 (see, e.g., Sheridan et al., Science,277:818-821 (1997); Pan et al., Science, 277:815-818 (1997), WO98/51793;WO98/41629; Screaton et al., Curr. Biol., 7:693-696 (1997); Walczak etal., EMBO J., 16:5386-5387 (1997); Wu et al., Nature Genetics,17:141-143 (1997); WO98/35986; EP870,827; WO98/46643; WO99/02653;WO99/09165; WO99/11791; US 2002/0072091; US 2002/0098550; U.S. Pat. No.6,313,269; US 2001/0010924; US 2003/01255540; US 2002/0160446, US2002/0048785; U.S. Pat. Nos. 6,342,369; 6,569,642, 6,072,047, 6,642,358;6,743,625) are used interchangeably, and include variants, isoforms,species homologs of human DR5, and analogs having at least one commonepitope with DR5. The complete human DR5 sequence can be found underGenBank Accession No. AAC01565.1 (SEQ ID NO: 26).

DR5 is a member of the tumor necrosis factor (TNF) receptor superfamily.TNF ligands are known to be among the most pleiotropic cytokines,inducing a large number of cellular responses, including cytotoxicity,anti-viral activity, immunoregulatory activities, and thetranscriptional regulation of several genes. Cellular responses toTNF-family ligands include not only normal physiological responses, butalso diseases associated with increased apoptosis or the inhibition ofapoptosis. Apoptosis (i.e., programmed cell death) is a physiologicalmechanism involved in the deletion of peripheral T lymphocytes of theimmune system, and its dysregulation can lead to a number of differentpathogenic processes. Diseases associated with increased cell survival,or the inhibition of apoptosis, include cancers, autoimmune disorders,viral infections, inflammation, graft versus host disease, acute graftrejection, and chronic graft rejection. Diseases associated withincreased apoptosis include AIDS, neurodegenerative disorders,myelodysplastic syndromes, ischemic injury, toxin-induced liver disease,septic shock, cachexia and anorexia.

The death receptors are characterized by their cysteine rich domains inthe extracellular region and death domains (DD) in the intracellularregion. Death domain endows death receptor with function of inducingcell death by apoptosis, but sometime it also mediates other signals.Tumor necrosis factor-related apoptosis-inducing ligand, TRAIL (Wiley SR, Schooley K, Smolak P, et al., Immunity, 1995, 3:673-682) incombination with its death domains triggers two cell death signalingpathways, i.e., death receptor pathway and mitochondrion pathway, tokill various tumor cells, but is nontoxic to most normal human cells.

Five TRAIL receptors, i.e., DR4 (death receptor 4 or named as TRAIL-R1),DR5, DcR1 (decoy receptor 1 or named as TRID/TRAIL-R3/LIT), DcR2(TRAIL-R4 or named as TRUNDD), and osteoprotegerin (OPG), have beenidentified. Like DR4, DR5 contains three cysteine-rich domains in itsextracellular portion and a single cytoplasmic death domain and becapable of signaling apoptosis upon ligand binding (or upon binding amolecule, such as an agonist (e.g., antibody), which mimics the activityof the ligand).

The term “agonist” as used with reference to DR5 refers to any moleculethat partially or fully enhances, stimulates or activates one or morebiological activities of DR5, in vitro, in situ, or in vivo. Examples ofsuch biological activities binding of Apo2L/TRAIL to DR5, includeapoptosis as well as those further reported in the literature. DR5agonists may function in a direct or indirect manner. For example, theDR5 agonist may function to partially or fully enhance, stimulate oractivate one or more biological activities of DR5, in vitro, in situ, orin vivo as a result of its direct binding to DR5, which causes receptoractivation or signal transduction. The DR5 agonist may also functionindirectly to partially or fully enhance, stimulate or activate one ormore biological activities of DR5, in vitro, in situ, or in vivo as aresult of, e.g., stimulating another effector molecule which then causesDR5 activation or signal transduction. It is contemplated that anagonist may act as an enhancer molecule which functions indirectly toenhance or increase DR5 activation or activity.

A DR5 agonist may be any molecule that directly or indirectly enhancesthe activity of DR5 and reduces tumor growth, whether on its own or incombination with another treatment, such as a PD-1 antagonist. ExemplaryDR5 agonists include DR5 binding scaffolds, such as anti-DR5 antibodies(“DR5 antibodies”), e.g., chimeric, humanized or fully human antibodies,an antigen binding portion thereof, or molecules that are based on orderived from any of these. DR5 agonists may also be non-antibodyproteins. DR5 agonist also include DR5 ligands, e.g., TRAIL andmolecules that are derived from or based on TRAIL.

A DR5 agonist may be monovalent or multivalent. In certain embodiments,a DR5 agonist is bivalent, trivalent, tetravalent, or binds to 5, 6, 7,8, 9, 10 or more DR5 epitopes, which may be the same or different DR5epitopes. In certain embodiments, a DR5 agonist is a multivalentmonospecific DR5 binding scaffold, e.g., a protein comprising a DR5binding scaffold that comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10 ormore regions that specifically bind to the same DR5 epitope, whichbinding regions may be composed of the same or a different amino acidsequence. For example, a DR5 agonist may be a DR5 binding scaffoldcomprising 2, 3, 4, 5, 6, 7, 8, 9, 10 or more repeats of the same DR5binding region. Multimeric DR5 binding scaffolds are described, e.g., inWO2009/058379, WO2011/130328, WO2010/042890 and WO2011/098520.

In certain embodiments, a DR5 agonist binds specifically to DR5, butdoes not bind significantly or specifically to other members of the TNFreceptor superfamily, such as DR4. In other embodiments, a DR5 agonistbinds specifically to DR5 and DR4.

For example, in one embodiment, the DR5 agonist is a recombinant humanTRAIL (TNF-related apoptosis-inducing ligand), e.g., Dulanermin (alsoknown as AMG-951; available from Amgen/Genentech).

In another embodiment, the DR5 agonist is an antibody, e.g., an antibodythat binds to human DR5 with a K_(D) of 10⁻⁷ M, 5×10⁻⁸ M, 10⁻⁸ M, 5×10⁻⁹M, 10⁻⁹ M, 5×10⁻¹⁰ M, 10⁻¹⁰ M or less, wherein the antibody inhibitstumor growth and/or induces apoptosis of tumor cells. Numerousantibodies binding to human DR5 are known in the art and some of themhave been used in clinical trials. Any of these antibodies may be usedin combination with a PD-1 antagonist, provided that their combinationresults in inhibition of tumor growth or reduction in tumor size, e.g.,in a subject having cancer. Exemplary antibodies that bind specificallyto human DR5 include Conatumumab (a hTRAILR2-specific antibody alsoknown as AMG655; available from Amgen), Drozitumab (a hTRAILR2-specificantibody also known as Apomab, DAB4, and PR095780; available fromGenentech), Lexatumumab (a hTRAILR2-specific antibody also known asHGS-ETR2; available from HGS/Kirin), Tigatuzumab (a humanizedTRAILR2-specific antibody also known as CS-1008 and TRA-8; availablefromDaiichi Sankyo), HGSTR2J (a hTRAILR2-specific antibody also known asKMTRS), or LBY-135 (a TRAILR2-specific Ab; available from Novartis)(see, e.g., Ashkenazi et al., Journal of Clinical Investigation 2008;118:1979-90). In one embodiment, the DR5 agonist is a bispecific deathreceptor agonist antibody, see, e.g., WO2011/039126; available fromRoche Glycart). In another embodiment, the DR5 agonist is an antibodyconjugated to targeting peptides or a cytotoxin, Fc-human TRAIL ligandfusion (see, e.g., WO2011/039126; available from Roche Glycart). Inanother embodiment, the DR5 agonist is a high affinity Fc-polypeptides(see, e.g., WO2011/143614; available from Amgen).

In another embodiment, the DR5 agonist is a multivalent agent, such asTAS266 (a tetrameric nanobody agonist targeting DR5, see, e.g.,WO2011/098520 and Cancer Research 2012; 72:Supplement 1; Abstract 3852;available from Novartis and Ablynx), multimeric Tn3 protein (see, e.g.,WO2009/058379, WO2011/130328, and Cancer Research 2012; 72:Supplement 1;Abstract 239; available from Medimmune), a multimer (e.g., a polypeptideconstruct with trimerizing domain and a polypeptide that binds DR5; seeWO2010/042890; available from Anaphore).

Agents, which compete for binding to DR5 with any of the exemplaryagents listed herein, and which inhibit tumor growth or reduces tumorsize may also be used. Antibodies having VH and VL chains comprising anamino acid sequence that is at least 90%, 95%, 98% or 99% identical tothose of any of the anti-DR5 antibodies listed herein may be used. Incertain embodiments of the methods described herein, a DR5 agonist isreplaced with a DR4 agonist. Thus, in certain embodiments, a subjecthaving cancer is treated with a combination of a DR4 agonist and a PD-1antagonist. Generally, any agent that induces apoptosis in tumor cellscan be combined with a PD-1 antagonist for treating cancer. In certainembodiments, an apoptosis inducing agent is an agent that bindsspecifically to DR5 and DR4, such as TRAIL or an agent that mimicsTRAIL. An exemplary DR4 agonist is Mapatumumab (HGS-ETR1), which hasbeen used in phase 2 clinical trials.

IV. Compositions

In one aspect, the present invention provides composition comprising aPD-1 antagonist and a DR5 agonist (e.g., formulated together in a singlecomposition or separately formulated). In one embodiment, thecomposition comprises a PD-1 antagonist and a DR5 agonist, wherein (a)the PD-1 antagonist is an anti-PD-1 antibody comprising the CDR1, CDR2and CDR3 domains in a heavy chain variable region having the sequenceset forth in SEQ ID NO: 13, and the CDR1, CDR2 and CDR3 domains in alight chain variable region having the sequence set forth in SEQ ID NO:15; and (b) the DR5 agonist is an antibody. In another embodiment, thecomposition comprises a PD-1 antagonist and a DR5 agonist, wherein (a)the PD-1 antagonist is an anti-PD-L1 antibody comprising the CDR1, CDR2and CDR3 domains in a heavy chain variable region having the sequenceset forth in SEQ ID NO: 1, and the CDR1, CDR2 and CDR3 domains in alight chain variable region having the sequence set forth in SEQ ID NO:3 and (b) the DR5 agonist is an antibody.

Pharmaceutical compositions suitable for administration to humanpatients are typically formulated for parenteral administration, e.g.,in a liquid carrier, or suitable for reconstitution into liquid solutionor suspension for intravenous administration.

In general, such compositions typically comprise a pharmaceuticallyacceptable carrier. As used herein, the term “pharmaceuticallyacceptable” means approved by a government regulatory agency or listedin the U.S. Pharmacopeia or another generally recognized pharmacopeiafor use in animals, particularly in humans. The term “carrier” refers toa diluent, adjuvant, excipient, or vehicle with which the compound isadministered. Such pharmaceutical carriers can be sterile liquids, suchas water and oils, including those of petroleum, animal, vegetable orsynthetic origin, such as peanut oil, soybean oil, mineral oil, sesameoil, glycerol polyethylene glycol ricinoleate, and the like. Water oraqueous solution saline and aqueous dextrose and glycerol solutions maybe employed as carriers, particularly for injectable solutions. Liquidcompositions for parenteral administration can be formulated foradministration by injection or continuous infusion. Routes ofadministration by injection or infusion include intravenous,intraperitoneal, intramuscular, intrathecal and subcutaneous.

For oral use, the pharmaceutical compositions of the present invention,may be administered, for example, in the form of tablets or capsules,powders, dispersible granules, or cachets, or as aqueous solutions orsuspensions. In the case of tablets for oral use, carriers which arecommonly used include lactose, corn starch, magnesium carbonate, talc,and sugar, and lubricating agents such as magnesium stearate arecommonly added. For oral administration in capsule form, useful carriersinclude lactose, corn starch, magnesium carbonate, talc, and sugar. Whenaqueous suspensions are used for oral administration, emulsifying and/orsuspending agents are commonly added.

In addition, sweetening and/or flavoring agents may be added to the oralcompositions. For intramuscular, intraperitoneal, subcutaneous andintravenous use, sterile solutions of the active ingredient(s) areusually employed, and the pH of the solutions should be suitablyadjusted and buffered. For intravenous use, the total concentration ofthe solute(s) should be controlled in order to render the preparationisotonic.

For preparing suppositories according to the invention, a low meltingwax such as a mixture of fatty acid glycerides or cocoa butter is firstmelted, and the active ingredient is dispersed homogeneously in the wax,for example by stirring. The molten homogeneous mixture is then pouredinto conveniently sized molds and allowed to cool and thereby solidify.

Liquid preparations include solutions, suspensions and emulsions. Suchpreparations are exemplified by water or water/propylene glycolsolutions for parenteral injection. Liquid preparations may also includesolutions for intranasal administration.

Aerosol preparations suitable for inhalation may include solutions andsolids in powder form, which may be in combination with apharmaceutically acceptable carrier, such as an inert compressed gas.

Also included are solid preparations which are intended for conversion,shortly before use, to liquid preparations for either oral or parenteraladministration. Such liquid forms include solutions, suspensions andemulsions.

V. Patient Populations

Provided herein are effective methods for treating cancer in a patient,e.g., using a combination of a DR5 agonist and PD-1 antagonist. In oneembodiment, the patient suffers from a cancer selected from the groupconsisting of leukemia, lymphoma, blastoma, carcinoma and sarcoma. Inanother embodiment, the patient suffers from a cancer selected from thegroup consisting of chronic myeloid leukemia, acute lymphoblasticleukemia, Philadelphia chromosome positive acute lymphoblastic leukemia(Ph+ ALL), squamous cell carcinoma, small-cell lung cancer, non-smallcell lung cancer, glioma, gastrointestinal cancer, renal cancer, ovariancancer, liver cancer, colorectal cancer, endometrial cancer, kidneycancer, prostate cancer, thyroid cancer, neuroblastoma, pancreaticcancer, glioblastoma multiforme, cervical cancer, stomach cancer,bladder cancer, hepatoma, breast cancer, colon carcinoma, and head andneck cancer, gastric cancer, germ cell tumor, pediatric sarcoma,sinonasal natural killer, multiple myeloma, acute myelogenous leukemia(AML), and chronic lymphocytic leukemia (CML).

VI. Additional Agents/Therapies

The combinations of the present invention (e.g., PD-1 antagonist incombination with DR5 agonist) may also be used in conjunction with otherwell known therapies that are selected for their particular usefulnessagainst the cancer that is being treated. Combinations of the instantinvention may alternatively be used sequentially with knownpharmaceutically acceptable agent(s) when inappropriate.

For example, the PD-1 antagonists and DR5 agonists described herein canfurther be used in combination (e.g., simultaneously or separately) withan additional treatment, such as irradiation, chemotherapy (e.g., usingcamptothecin (CPT-11), 5-fluorouracil (5-FU), cisplatin, doxorubicin,irinotecan, paclitaxel, gemcitabine, cisplatin, paclitaxel, doxorubicin,5-fu, or camptothecin+apo21/TRAIL (a 6× combo)), one or more proteasomeinhibitors (e.g., bortezomib or MG132), one or more Bcl-2 inhibitors(e.g., BH3I-2′ (bcl-xl inhibitor), AT-101 (R-(−)-gossypol derivative),ABT-263 (small molecule), GX-15-070 (obatoclax), or MCL-1 (myeloidleukemia cell differentiation protein-1) antagonists), iAP (inhibitor ofapoptosis protein) antagonists (e.g., smac7, smac4, small molecule smacmimetic, synthetic smac peptides (see Fulda et al., Nat Med 2002;8:808-15), ISIS23722 (LY2181308), or AEG-35156 (GEM-640)), HDAC (histonedeacetylase) inhibitors, anti-CD20 antibodies (e.g., rituximab),angiogenesis inhibitors (e.g., bevacizumab), anti-angiogenic agentstargeting VEGF and VEGFR, synthetic triterpenoids (see Hyer et al.,Cancer Research 2005; 65:4799-808), c-FLIP (cellular FLICE-inhibitoryprotein) modulators (e.g., natural and synthetic ligands of PPARγ(peroxisome proliferator-activated receptor 7), 5809354 or 5569100),kinase inhibitors (e.g., Sorafenib), and/or genotoxic drugs.

The PD-1 antagonists and DR5 agonists described herein can further beused in combination with one or more anti-proliferative cytotoxicagents. Classes of compounds that may be used as anti-proliferativecytotoxic agents include, but are not limited to, the following:

Alkylating agents (including, without limitation, nitrogen mustards,ethylenimine derivatives, alkyl sulfonates, nitrosoureas and triazenes):Uracil mustard, Chlormethine, Cyclophosphamide (CYTOXAN™) fosfamide,Melphalan, Chlorambucil, Pipobroman, Triethylenemelamine,Triethylenethiophosphoramine, Busulfan, Carmustine, Lomustine,Streptozocin, Dacarbazine, and Temozolomide.

Antimetabolites (including, without limitation, folic acid antagonists,pyrimidine analogs, purine analogs and adenosine deaminase inhibitors):Methotrexate, 5-Fluorouracil, Floxuridine, Cytarabine, 6-Mercaptopurine,6-Thioguanine, Fludarabine phosphate, Pentostatine, and Gemcitabine.

Suitable anti-proliferative agents for use in the methods of theinvention, include, without limitation, taxanes, paclitaxel (paclitaxelis commercially available as TAXOL®), docetaxel, discodermolide (DDM),dictyostatin (DCT), Peloruside A, epothilones, epothilone A, epothiloneB, epothilone C, epothilone D, epothilone E, epothilone F,furanoepothilone D, desoxyepothilone B1, [17]-dehydrodesoxyepothilone B,[18]dehydrodesoxyepothilones B, C12,13-cyclopropyl-epothilone A, C6-C8bridged epothilone A, trans-9,10-dehydroepothilone D,cis-9,10-dehydroepothilone D, 16-desmethylepothilone B, epothilone B10,discoderomolide, patupilone (EPO-906), KOS-862, KOS-1584, ZK-EPO,ABJ-789, XAA296A (Discodermolide), TZT-1027 (soblidotin), ILX-651(tasidotin hydrochloride), Halichondrin B, Eribulin mesylate (E-7389),Hemiasterlin (HTI-286), E-7974, Cyrptophycins, LY-355703, Maytansinoidimmunoconjugates (DM-1), MKC-1, ABT-751, T1-38067, T-900607, SB-715992(ispinesib), SB-743921, MK-0731, STA-5312, eleutherobin,17beta-acetoxy-2-ethoxy-6-oxo-B-homo-estra-1,3,5(10)-trien-3-ol,cyclostreptin, isolaulimalide, laulimalide,4-epi-7-dehydroxy-14,16-didemethyl-(+)-discodermolides, andcryptothilone 1, in addition to other microtubuline stabilizing agentsknown in the art.

In cases where it is desirable to render aberrantly proliferative cellsquiescent in conjunction with or prior to treatment with thechemotherapeutic methods of the invention, hormones and steroids(including synthetic analogs), such as 17a-Ethinylestradiol,Diethylstilbestrol, Testosterone, Prednisone, Fluoxymesterone,Dromostanolone propionate, Testolactone, Megestrolacetate,Methylprednisolone, Methyl-testosterone, Prednisolone, Triamcinolone,Chlorotrianisene, Hydroxyprogesterone, Aminoglutethimide, Estramustine,Medroxyprogesteroneacetate, Leuprolide, Flutamide, Toremifene, ZOLADEX™,can also be administered to the patient. When employing the methods orcompositions of the present invention, other agents used in themodulation of tumor growth or metastasis in a clinical setting, such asantimimetics, can also be administered as desired.

Methods for the safe and effective administration of chemotherapeuticagents are known to those skilled in the art. In addition, theiradministration is described in the standard literature. For example, theadministration of many of the chemotherapeutic agents is described inthe Physicians' Desk Reference (PDR), e.g., 1996 edition (MedicalEconomics Company, Montvale, N.J. 07645-1742, USA); the disclosure ofwhich is incorporated herein by reference thereto.

The chemotherapeutic agent(s) and/or radiation therapy can beadministered according to therapeutic protocols well known in the art.It will be apparent to those skilled in the art that the administrationof the chemotherapeutic agent(s) and/or radiation therapy can be varieddepending on the disease being treated and the known effects of thechemotherapeutic agent(s) and/or radiation therapy on that disease.Also, in accordance with the knowledge of the skilled clinician, thetherapeutic protocols (e.g., dosage amounts and times of administration)can be varied in view of the observed effects of the administeredtherapeutic agents on the patient, and in view of the observed responsesof the disease to the administered therapeutic agents.

VII. Treatment Protocols

Suitable treatment protocols for treating cancer in a patient include,for example, administering to the patient an effective amount of a PD-1antagonist (e.g., antibody) and a DR5 agonist (e.g., antibody).

As used herein, adjunctive or combined administration(co-administration) includes simultaneous administration of theantagonist and agonist in the same or different dosage form, or separateadministration of the antagonist and agonist (e.g., sequentialadministration). Thus, the PD-1 antagonist (e.g., antibody) and DR5agonist (e.g., antibody) can be simultaneously administered in a singleformulation. Alternatively, the PD-1 antagonist and DR5 agonist can beformulated for separate administration and are administered concurrentlyor sequentially.

For example, the PD-1 antagonist can be administered first followed by(e.g., immediately followed by) the administration of the DR5 agonist,or vice versa. In one embodiment, the PD-1 antagonist is administeredprior to administration of the DR5 agonist. In one embodiment, the DR5agonist is administered prior to administration of the PD-1 antagonist.Such concurrent or sequential administration preferably results in boththe agonist and antagonist being simultaneously present in treatedpatients. In another embodiment, the DR5 agonist and the PD-1 antagonistare administered simultaneously.

In an exemplary treatment, a subject is dosed with a single dose of aDR5 agonist and at least 2 doses of a PD-1 antagonist, e.g., ananti-PD-1 or anti-PD-L1 antibody. In certain embodiment, a subjectreceives a single dose of a DR5 agonist and at least 2, 3, 4, 5, or moredoses of a PD-1 antagonist. The multiple doses of PD-1 antagonist may beprovided as one dose per day, one dose every 2 days, one dose every 3days, one dose every 4 days, one dose every 5 days or less frequently.In certain embodiments, in which a PD-1 antagonist is provided as 1 doseevery 1, 2, 3, 4, 5 or more days, the single dose of DR5 agonist may beprovided on a day on which the PD-1 antagonist is provided or on a dayon which it is not provided. The total number of doses of PD-1antagonist may be 2, 3, 4, 5, 6, 7, 8, 9, 10 or more.

In certain embodiments, multiple (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 ormore) doses of a DR5 agonist and multiple (e.g., 2, 3, 4, 5, 6, 7, 8, 9,10 or more) doses of a PD-1 antagonist are administered to a subject inneed of treatment. Administration of the DR5 agonist and the PD-1antagonist may be on the same day, or alternatively, the DR5 antagonistmay be administered 1 or more days before or after the PD-1 antagonist.

Administrations of a DR5 agonist and a PD-1 antagonist may also be doneweekly or monthly, in which regimen, they may be administered on thesame day (e.g., simultaneously), or one after the other (e.g., one ormore days before or after one another)

In one embodiment, the dose of the PD-1 antagonist and/or DR5 agonist isvaried over time. For example, the PD-1 antagonist and/or DR5 agonistmay be initially administered at a high dose and may be lowered overtime. In another embodiment, the PD-1 antagonist and/or DR5 agonist isinitially administered at a low dose and increased over time.

In another embodiment, the amount of the PD-1 antagonist and/or DR5agonist administered is constant for each dose. In another embodiment,the amount of the PD-1 antagonist and/or DR5 agonist varies with eachdose. For example, the maintenance (or follow-on) dose of the antagonistand/or agonist can be higher or the same as the loading dose which isfirst administered. In another embodiment, the maintenance dose of theantagonist and/or agonist can be lower or the same as the loading dose.A clinician may utilize preferred dosages as warranted by the conditionof the patient being treated. The dose of may depend upon a number offactors, including stage of disease, etc. The specific dose that shouldbe administered based upon the presence of one or more of such factorsis within the skill of the artisan. Generally, treatment is initiatedwith smaller dosages which are less than the optimum dose of thecompound. Thereafter, the dosage is increased by small amounts until theoptimum effect under the circumstances is reached. For convenience, thetotal daily dosage may be divided and administered in portions duringthe day if desired. Intermittent therapy (e.g., one week out of threeweeks or three out of four weeks) may also be used.

In one embodiment, the DR5 agonist (e.g., antibody) is administered at adose of 0.1, 0.3, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg/kg body weight. Inanother embodiment, the PD-1 antagonist (e.g., antibody) is administeredat a dose of 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg/kg body weight.Generally, 200 μg/mouse is approximately 10 mg/kg and 100 μg/mouse isapproximately 5 mg/kg. Therefore, based on the experiments describedherein, one or more doses of 1-20 mg/kg body weight, 1-10 mg/kg bodyweight, 5-20 mg/kg body weight or 5-10 mg/kg body weight of a DR5agonist and PD-1 antagonist may be administered to a subject. In certainembodiments, a dose of 0.3 mg/kg to 10 mg/kg body weight of a DR5agonist is used and a dose of at least 1 mg/kg, e.g., 1-100 mg/kg bodyweight of a PD-1 antagonist is used.

VIII. Outcomes

Patients, e.g., humans, treated according to the methods disclosedherein preferably experience improvement in at least one sign of cancer.In one embodiment, improvement is measured by a reduction in thequantity and/or size of measurable tumor lesions. In another embodiment,lesions can be measured on chest x-rays or CT or MRI films. In anotherembodiment, cytology or histology can be used to evaluate responsivenessto a therapy.

In one embodiment, the patient treated exhibits a reduction in size of atumor, reduction in number of metastasic lesions over time, completeresponse, partial response, and stable disease. In another embodiment,the patient treated experiences tumor shrinkage and/or decrease ingrowth rate, i.e., suppression of tumor growth. In another embodiment,unwanted cell proliferation is reduced or inhibited. In yet anotherembodiment, one or more of the following can occur: the number of cancercells can be reduced; tumor size can be reduced; cancer cellinfiltration into peripheral organs can be inhibited, retarded, slowed,or stopped; tumor metastasis can be slowed or inhibited; tumor growthcan be inhibited; recurrence of tumor can be prevented or delayed; oneor more of the symptoms associated with cancer can be relieved to someextent.

In another embodiment, the methods of treatment produce a comparableclinical benefit rate (CBR=CR (complete response), PR (partial response)or SD (stable disease)≥6 months) better than that achieved by a PD-1(e.g., antibody) or DR5 agonist (e.g., antibody) alone. In otherembodiments, the improvement of clinical benefit rate is about 10%, 20%,30%, 40%, 50%, 60%, 70%, 80% or more, e.g., compared to treatment with aPD-1 antagonist or DR5 agonist alone or relative to tumor growth on thefirst day of treatment or immediately before initiation of treatment.

In another embodiment, administration of a PD-1 antagonist and a DR5agonist results in at least a three-fold reduction (e.g., a 3.5-foldreduction) in tumor volume, e.g., relative to treatment with the PD-1antagonist or the DR5 agonist alone or relative to tumor growth on thefirst day of treatment or immediately before initiation of treatment.

In a further embodiment, administration of a PD-1 antagonist and a DR5agonist results in tumor growth inhibition of at least 80%, e.g.,relative to treatment with the PD-1 antagonist or DR5 agonist alone orrelative to tumor growth on the first day of treatment or immediatelybefore initiation of treatment.

In certain embodiments, administration of a PD-1 antagonist and a DR5agonist reduces tumor mass by at least 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, 95%, 99% relative to the tumor mass prior to initiationof the treatment or on the first day of treatment. In some embodiment,the tumor mass is no longer detectable following treatment as describedherein. In some embodiments, a subject is in partial or full remission.

IX. Kits and Unit Dosage Forms

Also provided herein are kits which include a pharmaceutical compositioncontaining (a) a PD-1 antagonist and (b) a DR5 agonist and apharmaceutically-acceptable carrier, in a therapeutically effectiveamount adapted for use in the preceding methods. In one embodiment, thePD-1 antagonist is an antibody (e.g., 5C4 or 12A4, respectively). Inanother embodiment, the DR5 agonist is an antibody. The kits optionallyalso can include instructions, e.g., comprising administrationschedules, to allow a practitioner (e.g., a physician, nurse, orpatient) to administer the composition contained therein to a patienthaving cancer. The kit also can include a syringe.

Optionally, the kits include multiple packages of the single-dosepharmaceutical compositions each containing an effective amount of thePD-1 antagonist and the DR5 agonist for a single administration inaccordance with the methods provided above. Instruments or devicesnecessary for administering the pharmaceutical composition(s) also maybe included in the kits. For instance, a kit may provide one or morepre-filled syringes containing an amount of the PD-1 antagonist and theDR5 agonist.

In one embodiment, the present invention provides a kit for treatingcancer in a patient, the kit comprising:

(a) a dose of a PD-1 antagonist;

(b) a dose of a DR5 agonist; and

(c) instructions for using the PD-1 antagonist and DR5 agonist in themethod of any one of claims 1-32. In another embodiment, the DR5 agonistis an antibody. In another embodiment, the PD-1 antagonist is anantibody. In particular embodiment, the PD-1 antagonist is an anti-PD-1antibody comprising the CDR1, CDR2 and CDR3 domains in a heavy chainvariable region having the sequence set forth in SEQ ID NO: 13, and theCDR1, CDR2 and CDR3 domains in a light chain variable region having thesequence set forth in SEQ ID NO: 15. In another particular embodiment,the PD-1 antagonist is an anti-PD-L1 antibody comprising antibodycomprises the CDR1, CDR2 and CDR3 domains in a heavy chain variableregion having the sequence set forth in SEQ ID NO: 1, and the CDR1, CDR2and CDR3 domains in a light chain variable region having the sequenceset forth in SEQ ID NO: 3.

The following examples are merely illustrative and should not beconstrued as limiting the scope of this disclosure in any way as manyvariations and equivalents will become apparent to those skilled in theart upon reading the present disclosure.

The contents of all references, Genbank entries, patents and publishedpatent applications cited throughout this application are expresslyincorporated herein by reference.

EXAMPLES Example 1 1. Materials and Methods

Animals

Ten to eleven-week-old female C57/BL6 mice (Harlan) were used in thestudies. Mice received food and water ad libitum and were maintained ina controlled environment according to Association for Assessment andAccreditation of Laboratory Animal Care (AAALAC) Internationalregulations. All animal studies have been approved by the appropriateethics committee and have therefore been performed in accordance withthe ethical standards laid down in the 1964 Declaration of Helsinki andits later amendments.

Antibodies

Anti-mouse PD-1 mAb (anti-mPD-1 mAb) clone 4H2, mouse IgG1 isotype wasproduced and purified by Bristol-Myers Squibb (Biologics Discovery, CA).Agonist anti-mouse DR5 mAb, clone MD5-1, hamster IgG isotype, waspurchased from BioxCell (West Lebanon, N.H.). Both antibodies werecertified to have <0.5 EU/mg endotoxin levels, >95% purity and <5% highmolecular weight species. Stock solutions of anti-mPD-1 mAb andanti-mDR5 antibody were kept at 4° C. prior to use. Dosing solutions ofanti-mPD-1 mAb and anti-mDR5 mAb were prepared in sterile phosphatebuffered saline (pH 7.0) and maintained at 4° C.

Anti-mPD-1 mAb was administered intraperitoneally at its optimal dose of10 mg/kg; anti-DR5 mAb at 5 mg/kg.

Tumor Model

The MC38 colon carcinoma tumor line used in this study was maintained invitro. Cell suspensions were implanted in the subcutaneous space of theflank of mice of female C57/BL6 mice (2.0×10⁶ MC-38 cells in 0.2 mLHanks Balanced Salt Solution).

Tumor size and body weights were measured twice weekly. Tumor size(measured as mm³) was calculated by multiplying the tumor length by thesquare of the tumor width divided by 2. Treatments were initiated whensubcutaneous tumors reached a median size of 200 mm³ (establishedmodel). Antitumor activity, defined as percentage tumor growthinhibition, was calculated with the formula % Tumor Growth Inhibition (%TGI)=100−[(Tt/To)/(Ct/Co)]/100−(Ct/Co), where Tt=median tumor size oftreated group at the end of treatment, To=median tumor size of treatedgroup at treatment initiation, Ct=median tumor size of control group atthe end of treatment, and Co=median tumor size at treatment initiation(see Table 3). Complete regressions were defined as absence ofmeasurable tumor mass for at least 2 tumor volume doubling times.

The tumor response endpoint was expressed as tumor growth delay (T-Cvalue), calculated as the difference in time (days) between the treated(T) and control (C) groups for the tumor to reach a predetermined targetsize. A delay in reaching target size by the treated groups of >1 timestumor volume doubling time was considered an active result. Therapeuticsynergy was defined as an antitumor effect in which the combination ofagents demonstrated significant superiority (p<0.05) relative to theactivity shown by each agent alone.

The antitumor effect of single dose anti-mouse DR5 mAb in combination ananti-PD-1 mAb given at various dose schedules was evaluated in MC-38(murine colon) tumor bearing mice. Six days post tumor implant, micewere sorted into eight groups of 7 mice with a mean tumor volume of 200mm³. Antibodies were administered according to the dosing schedulesdescried in Table 1.

TABLE 1 Dosing Schedule Treatment Group # mice 1: Control 7 2: Control +anti-PD-1 mAb; 200 ug/mouse; 7 Q4D × 3 dosing initiated day 6 3:Control + anti-PD-1 mAb; 200 ug/mouse; 7 Q4D × 3 dosing initiated day 84: Control + anti-PD-1 mAb; 200 ug/mouse; 7 Q4D × 3 dosing initiated day9 5: Anti-DR5 mAb; 100 ug/mouse; 7 QD + Control dosing initiated day 86: Anti-DR5 mAb; 100 ug/mouse dosed 7 day 8; QD + anti-PD-1 mAb; 200ug/mouse; Q4D × 3 dosed day 6 7: Anti-DR5 mAb; 100 ug/mouse dosed 7 day8; QD + anti-PD-1 mAb; 200 ug/mouse; Q4D × 3 dosed day 8 8: Anti-DR5mAb; 100 ug/mouse dosed 7 day 8; QD + anti-PD-1 mAb; 200 ug/mouse; Q4D ×3 dosed day 9 QD: One dose administered on only one day. Q4D × 3: Onedose administered every four days for a total of 3 doses.

The combination of DR5 mAb and PD-1 mAb were tested according to thethree schedules set forth in Table 2.

TABLE 2 Administration Schedules Group No.: Administration ScheduleGroup 6 DR5 mAb was administered 2 days after PD-1 mAb therapy. Group 7DR5 mAb and PD-1 mAb were administered on the same day. Group 8 DR5 mAbwas administered 1 day before PD-1 mAb therapy.

It was hypothesized that since PD-1 mab induces IFN-gamma, which intumors upregulates DR5 expression on tumor cells, it might beadvantageous to administer the DR5 mAb after administration of PD-1 mAbtherapy (Group 6). Alternatively, it was hypothesized that DR5 mAbinduces tumor cell death, which in turn will prime antitumor immuneresponses, and PD-1 mAb will subsequently expand the induced antitumorimmunity. To test this hypothesis, DR5 mAb was administered before PD-1mAb therapy (Group 8).

2. Results

As shown in Table 3, at least 80% tumor growth inhibition was achievedin mice treated with a combination of both the DR5 mAb and PD1 mAb.

TABLE 3 Tumor Responses Schedule % Complete Dose (days post RegressionsTreatment (mg/kg) implant) % TGI (#/total mice) Anti-PD-1 mAb 10 Day 6,10, 14 31  0 (0/7) Anti-PD-1 mAb 10 Day 8, 12, 16 14  0 (0/7) Anti-PD-1mAb 10 Day 9, 13, 17 −8  0 (0/7) Anti-DR5 mAb 5 Day 8 −4  0 (0/7)Anti-PD-1 mAb + 10 Day 6, 10, 14 96 43 Anti-DR5 mAb 5 Day 8 (3/7)Anti-PD-1 mAb + 10 Day 8, 12, 16 82 14 Anti-DR5 mAb 5 Day 8 (1/7)Anti-PD-1 mAb + 10 Day 9, 13, 17 87 43 Anti-DR5 mAb 5 Day 8 (3/7)Moreover, as shown in FIG. 1, the medium tumor volume (measured in mm³)in mice treated with a combination of the DR5 mAb and the PD-1 mAb wassignificantly reduced, compared to mice treated with a control or eitheragent alone. Specifically, there was about a 3.5 fold reduction (e.g.,at least a 3 fold reduction) in tumor volume in mice treated with boththe DR5 mAb and the PD-1 mAb, compared to mice treated with a control oreither agent alone. The tumor volume in individual mice is shown inFIGS. 2A-2H.

In sum, the combination of the DR5 mAb and the PD-1 mAb resulted inenhanced activity compared to the activity elicited by single agentsalone, independent of the schedule utilized. This synergy wasstatistically significant (p<0.05, Wilcoxon). Out of the 3administration schedules tested, a trend for better activity wasobserved in the groups which were treated with anti-mDR5 first or withanti-mPD-1 first. As shown in FIG. 3, the combination therapy waswell-tolerated (no significant body weight loss). In previous studies,significant body weight loss (>20%) was observed with multiple doses ofthe DR5 mAb alone or in combination.

Therefore, results from this study demonstrate that a combinationregimen that includes a single dose of anti-mDR5 mAb and multiple dosesof PD-1 mAb is well-tolerated and result in marked antitumor activity.

SEQ ID NO:  SEQUENCE 1 Heavy Chain Variable Region (VH) Amino AcidSequence Anti-PD-L1 mAb (12A4; 12A4 in WO 2007/005874 andU.S. Pat. No. 7,943,743) QVQLVQSGAEVKKPGSSVKVSCKTSGDTFSTYAISWVRQAPGQGLEWM GGIIPIFGKAHYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYFC ARKFHFVSGSPFGMDVWGQGTTVT VSS 2Heavy Chain Variable Region (VH) Nucleotide SequenceAnti-PD-L1 mAb (12A4; 12A4 in WO 2007/005874and U.S. Pat. No. 7,943,743) cag gtc cag ctg gtg cagtct ggg gct gag gtg aag aag cct ggg tcc tcg gtg aag gtc tcc tgc aag acttct gga gac acc ttc agc acc tat gct atc agc tgg gtg cga cag gcc cct ggacaa ggg ctt gag tgg atg gga ggg atc atc cct ata ttt ggt aaa gca cac tacgca cag aag ttc cag ggc aga gtc acg att acc gcg gac gaa tcc acg agc acagcc tac atg gag ctg agc agc ctg aga tct gag gac acg gcc gtg tat ttt tgtgcg aga aag ttt cac ttt gtt tcg ggg agc ccc ttc ggt atg gac gtc tgg ggccaa ggg acc acg gtc acc gtc tcc 3 Light Chain Variable Region(VL) Amino Acid Sequence Anti-PD-Ll mAb (12A4; 12A4 in WO 2007/005874and U.S. Pat. No. 7,943,743) EIVITQSPATLSLSPGERATL SCRASQSVSSYLAWYQQKPCQAPRLLIYDASNRATGIPARFS GSGSGTDFTLTISSLEPEDFA VYYCQQRSNWPTFGQGTKVEIK 4Light Chain Variable Region (VL) Nucleotide Sequence Anti-PD-L1mAb (12A4; 12A4 in WO 2007/005874 and U.S. Pat. No. 7,943,743)gaa att gtg ttg aca cag tct cca gcc acc ctg tct ttg tct cca ggg gaa agagcc acc ctc tcc tgc agg gcc agt cag agt gtt agc agc tac tta gcc tgg taccaa cag aaa cct ggc cag gct ccc agg ctc ctc atc tat gat gca tcc aac agggcc act ggc atc cca gcc agg ttc agt ggc agt ggg tct ggg aca gac ttc actctc acc atc agc agc cta gag cct gaa gat ttt gca gtt tat tac tgt cag cagcgt agc aac tgg ccg acg ttc ggc caa ggg acc aag gtg gaa atc aaa 5Heavy Chain CDR1 Amino Acid Sequence Anti-PD-L1 mAb (12A4; 12A4 inWO 2007/005874 and U.S. Pat. No. 7,943,743) TYAIS 6Heavy Chain CDR2 Amino Acid Sequence Anti-PD-L1 mAb (12A4; 12A4 inWO 2007/005874 and U.S. Pat. No. 7,943,743) GIIPIFGKAHYAQKFQ 7Heavy Chain CDR3 Amino Acid Sequence Anti-PD-L1 mAb (12A4; 12A4 inWO 2007/005874 and U.S. Pat. No. 7,943,743) KFHFVSGSPFGMDV 8Light Chain CDR1 Amino Acid Sequence Anti-PD-L1 mAb (12A4; 12A4 inWO 2007/005874 and U.S. Pat. No. 7,943,743) RASQSVSSYLA 9Light Chain CDR2 Amino Acid Sequence Anti-PD-L1 mAb (12A4; 12A4 inWO 2007/005874 and U.S. Pat. No. 7,943,743) DASNRAT 10Light Chain CDR3 Amino Acid Sequence Anti-PD-L1 mAb (12A4; 12A4 inWO 2007/005874 and U.S. Pat. No. 7,943,743) QQRSNWPT 11Heavy Chain Amino Acid Sequence Anti-PD-1 mAb (5C4 in WO 2006/121168)(variable region underlined; constant region bold)QVQLVESGGGVVQPGRSLRLDCKA SGITFSNSGMHWVRQAPGKGLEWVAVIWYDGSKRYYADSVKGRFTISR DNSKNTLFLQMNSLRAEDTAVYYC ATNDDYWGQGTLVTVSSASTKGPS VFPLAPCSRSTSESTAALGCLVKD YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTK TYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQ FNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQP REPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQK SLSLSLGK 12 Light Chain Amino AcidSequence Anti-PD-1 mAb (5C4 in WO 2006/121168) (variable regionunderlined; constant region bold) EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLII YDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPR TFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLINNFYPREA KVQWKVDNALQSGNSQESVTEQDSDKSTYSLSSTLTLSKADYEKHKVY ACEVTHQGLSSPVTKSFNRGEC 13 Heavy Chain VariableRegion (VH) Amino Acid Sequence Anti-PD-1 mAb (5C4 in WO 2006/121168)(SEQ ID NO: 4 from WO 2006/121168) QVQLVESGGGWQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGL EWVAVIWYDGSKRYYADSVKGR FTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTV SS 14 Heavy Chain Variable Region (VH) NucleotideSequence Anti-PD-1 mAb (5C4 in WO 2006/121168) (SEQ ID NO: 60 fromWO 2006/121168) cag gtg cag ctg gtg gag tct ggg gga ggc gtg gtccag cct ggg agg tcc ctg aga ctc gac tgt aaa gcg tct gga atc acc ttc agtaac tct ggc atg cac tgg gtc cgc cag gct cca ggc aag ggg ctg gag tgg gtggca gtt att tgg tat gat gga agt aaa aga tac tat gca gac tcc gtg aag ggccga ttc acc atc tcc aga gac aat tcc aag aac acg ctg ttt ctg caa atg aacagc ctg aga gcc gag gac acg gct gtg tat tac tgt gcg aca aac gac gac tactgg ggc cag gga acc ctg gtc acc gtc tcc tca 15Light Chain Variable Region (VL) Amino Acid Sequence Anti-PD-1 mAb(5C4 in WO 2006/121168) (SEQ ID NO: 11 from WO 2006/121168)EIVLTQSPATLSLSPGERATLSCR ASQSVSSYLAWYQQKPGQAPRLIIYDASNRATGIPARFSGSGSGTDFT LTISSLEPEDFAVYYCQQSSNWPR TFGQGTKVEIK 16Light Chain Variable Region (VL) Nucleotide Sequence Anti-PD-1 mAb(5C4 in WO 2006/121168) (SEQ ID NO: 67 from WO 2006/121168)gaa att gtg ttg aca cag tct cca gcc acc ctg tct ttg tct cca ggg gaa agagcc acc ctc tcc tgc agg gcc agt cag agt gtt agt agt tac tta gcc tgg taccaa cag aaa cct ggc cag gct ccc agg ctc ctc atc tat gat gca tcc aac agggcc act ggc atc cca gcc agg ttc agt ggc agt ggg tct ggg aca gac ttc actctc acc atc agc agc cta gag cct gaa gat ttt gca gtt tat tac tgt cag cagagt agc aac tgg cct cgg acg ttc ggc caa ggg acc aag gtg gaa atc aaa 17Heavy Chain CDR1 Amino Acid Sequence Anti-PD-1mAb (5C4 in WO 2006/121168) (SEQ ID NO: 18 from WO 2006/121168) NSGMH 18Heavy Chain CDR2 Amino Acid Sequence Anti-PD-1mAb (5C4 in WO 2006/121168) (SEQ ID NO: 25 from WO 2006/121168)VIWYDGSKRYYADSVKG 19 Heavy Chain CDR3 Amino Acid Sequence Anti-PD-1mAb (5C4 in WO 2006/121168) (SEQ ID NO: 32 from WO 2006/121168) NDDY 20Light Chain CDR1 Amino Acid Sequence Anti-PD-1 mAb(5C4 in WO 2006/121168) (SEQ ID NO: 39 from WO 2006/121168) RASQSVSSYLA21 Light Chain CDR2 Amino Acid Sequence Anti-PD-1 mAb (5C4 inWO 2006/121168) (SEQ ID NO: 46 from WO 2006/121168) DASNRAT 22Light Chain CDR3 Amino Acid Sequence Anti-PD-1 mAb (5C4 inWO 2006/121168) (SEQ ID NO: 53 from WO 2006/121168) QQSSNWPRT 23Complete PD-1 sequence (GenBank Accession No.: U64863)agtttccctt ccgctcacct ccgcctgagc agtggagaag gcggcactct ggtggggctgctccaggcat gcagatccca caggcgccct ggccagtcgt ctgggcggtg ctacaactgggctggcggcc aggatggttc ttagactccc cagacaggcc ctggaacccc cccaccttcttcccagccct gctcgtggtg accgaagggg acaacgccac cttcacctgc agcttctccaacacatcgga gagcttcgtg ctaaactggt accgcatgag ccccagcaac cagacggacaagctggccgc cttccccgag gaccgcagcc agcccggcca ggactgccgc ttccgtgtcacacaactgcc caacgggcgt gacttccaca tgagcgtggt cagggcccgg cgcaatgacagcggcaccta cctctgtggg gccatctccc tggcccccaa ggcgcagatc aaagagagcctgcgggcaga gctcagggtg acagagagaa gggcagaagt gcccacagcc caccccagcccctcacccag gccagccggc cagttccaaa ccctggtggt tggtgtcgtg ggcggcctgctgggcagcct ggtgctgcta gtctgggtcc tggccgtcat ctgctcccgg gccgcacgagggacaatagg agccaggcgc accggccagc ccctgaagga ggacccctca gccgtgcctgtgttctctgt ggactatggg gagctggatt tccagtggcg agagaagacc ccggagccccccgtgccctg tgtccctgag cagacggagt atgccaccat tgtctttcct agcggaatgggcacctcatc ccccgcccgc aggggctcag ccgacggccc tcggagtgcc cagccactgaggcctgagga tggacactgc tcttggcccc tctgaccggc ttccttggcc accagtgttctgcagaccct ccaccatgag cccgggtcag cgcatttcct caggagaagc aggcagggtgcaggccattg caggccgtcc aggggctgag ctgcctgggg gcgaccgggg ctccagcctgcacctgcacc aggcacagcc ccaccacagg actcatgtct caatgcccac agtgagcccaggcagcaggt gtcaccgtcc cctacaggga gggccagatg cagtcactgc ttcaggtcctgccagcacag agctgcctgc gtccagctcc ctgaatctct gctgctgctg ctgctgctgctgctgctgcc tgcggcccgg ggctgaaggc gccgtggccc tgcctgacgc cccggagcctcctgcctgaa cttgggggct ggttggagat ggccttggag cagccaaggt gcccctggcagtggcatccc gaaacgccct ggacgcaggg cccaagactg ggcacaggag tgggaggtacatggggctgg ggactcccca ggagttatct gctccctgca ggcctagaga agtttcagggaaggtcagaa gagctcctgg ctgtggtggg cagggcagga aacccctccc acctttacacatgcccaggc agcacctcag gccctttgtg gggcagggaa gctgaggcag taagcgggcaggcagagctg gaggcctttc aggccagcca gcactctggc ctcctgccgc cgcattccaccccagcccct cacaccactc gggagaggga catcctacgg tcccaaggtc aggagggcagggctggggtt gactcaggcc cctcccagct gtggccacct gggtgttggg agggcagaagtgcaggcacc tagggccccc catgtgccca ccctgggagc tctccttgga acccattcctgaaattattt aaaggggttg gccgggctcc caccagggcc tgggtgggaa ggtacaggcgttcccccggg gcctagtacc cccgcgtggc ctatccactc ctcacatcca cacactgcacccccactcct ggggcagggc caccagcatc caggcggcca gcaggcacct gagtggctgggacaagggat cccccttccc tgtggttcta ttatattata attataatta aatatgagag catgct24 Human PD-L1 amino acid sequence-isoform a precursor (GenBankAccession No. NP_054862.1) MRIFAVFIFM TYWHLLNAFT VTVPKDLYVV EYGSNMTIECKFPVEKQLDL AALIVYWEME DKNIIQFVHG EEDLKVQHSS YRQRARLLKD QLSLGNAALQITDVKLQDAG VYRCMISYGG ADYKRITVKV NAPYNKINQR ILVVDPVTSE HELTCQAEGYPKAEVIWTSS DHQVLSGKTT TTNSKREEKL FNVTSTLRIN TTTNEIFYCT FRRLDPEENHTAELVIPELP LAHPPNERTH LVILGAILLC LGVALTFIFR LRKGRMMDVK KCGIQDTNSKKQSDTHLEET 25 Human PD-L1 amino acid sequence-isoform b precursor(GenBank Accession No. NP_001254635.1) MRIFAVFIFM TYWHLLNAPYNKINQRILVV DPVTSEHELT CQAEGYPKAE VIWTSSDHQV LSGKTTTTNS KREEKLFNVTSTLRINTTTN EIFYCTFRRL DPEENHTAEL VIPELPLAHP PNERTHLVIL GAILLCLGVALTFIFRLRKG RMMDVKKCGI QDTNSKKQSD THLEET 26 Human DR5 amino acidsequence (GenBank Accession No. AAC01565.1) MEQRGQNAPA ASGARKRHGPGPREARGARP GLRVPKTLVL VVAAVLLLVS AESALITQQD LAPQQRVAPQ QKRSSPSEGLCPPGHHISED GRDCISCKYG QDYSTHWNDL LFCLRCTRCD SGEVELSPCT TTRNTVCQCEEGTFREEDSP EMCRKCRTGC PRGMVKVGDC TPWSDIECVH KESGIIIGVT VAAVVLIVAVFVCKSLLWKK VLPYLKGICS GGGGDPERVD RSSQRPGAED NVLNEIVSIL QPTQVPEQEMEVQEPAEPTG VNMLSPGESE HLLEPAEAER SQRRRLLVPA NEGDPTETLR QCFDDFADLVPFDSWEPLMR KLGLMDNEIK VAKAEAAGHR DTLYTMLIKW VNKTGRDASV HTLLDALETLGERLAKQKIE DHLLSSGKFM YLEGNADSAM S

1-30. (canceled)
 31. A method of treating cancer in a subject, themethod comprising administering to the subject an effective amount of aPD-1 antagonist and a DR4 or DR5 agonist.
 32. The method of claim 31,wherein the PD-1 antagonist and the DR4 or DR5 agonist are selected fromthe group consisting of a ligand, antibody, and multivalent agent. 33.The method of claim 32, wherein the DR5 agonist is an antibody selectedfrom the group consisting of Lexatumumab, Tigatuzumab, Conatumumab,Drozitumab, HGSTR2J/KMTRS, and LBY-135.
 34. The method of claim 32,wherein the DR5 agonist is a multivalent agent comprising TAS266. 35.The method of claim 31, wherein the PD-1 antagonist is an anti-PD-1antibody comprising the CDR1, CDR2 and CDR3 domains in a heavy chainvariable region having the sequence set forth in SEQ ID NO: 13, and theCDR1, CDR2 and CDR3 domains in a light chain variable region having thesequence set forth in SEQ ID NO:
 15. 36. The method of claim 31, whereinthe PD-1 antagonist is an anti-PD-1 antibody comprising: (a) a heavychain variable region CDR1 having the sequence set forth in SEQ ID NO:17; (b) a heavy chain variable region CDR2 having the sequence set forthin SEQ ID NO: 18; (c) a heavy chain variable region CDR3 having thesequence set forth in SEQ ID NO: 19; (d) a light chain variable regionCDR1 having the sequence set forth in SEQ ID NO: 20; (e) a light chainvariable region CDR2 having the sequence set forth in SEQ ID NO: 21; and(f) a light chain variable region CDR3 having the sequence set forth inSEQ ID NO:
 22. 37. The method of claim 31, wherein the PD-1 antagonistis an anti-PD-1 antibody comprising heavy and light chain variableregions having the sequences set forth in SEQ ID NOs: 13 and 15,respectively.
 38. The method of claim 31, wherein the PD-1 antagonist isan anti-PD-1 antibody comprising heavy and light chains having thesequences as set forth in SEQ ID NOs: 11 and 12, respectively.
 39. Themethod of claim 31, wherein the PD-1 antagonist is an anti-PD-L1antibody comprising the CDR1, CDR2 and CDR3 domains in a heavy chainvariable region having the sequence set forth in SEQ ID NO: 1, and theCDR1, CDR2 and CDR3 domains in a light chain variable region having thesequence set forth in SEQ ID NO:
 3. 40. The method of claim 31, whereinthe PD-1 antagonist is an anti-PD-L1 antibody comprising: (a) a heavychain variable region CDR1 having the sequence set forth in SEQ ID NO: 5(b) a heavy chain variable region CDR2 having the sequence set forth inSEQ ID NO: 6; (c) a heavy chain variable region CDR3 having the sequenceset forth in SEQ ID NO: 7; (d) a light chain variable region CDR1 havingthe sequence set forth in SEQ ID NO: 8; (e) a light chain variableregion CDR2 having the sequence set forth in SEQ ID NO: 9; and (f) alight chain variable region CDR3 having the sequence set forth in SEQ IDNO:
 10. 41. The method of claim 31, wherein the PD-1 antagonist is ananti-PD-L1 antibody comprising heavy and light chain variable regionshaving the sequences set forth in SEQ ID NOs: 1 and 3, respectively. 42.The method of claim 31, wherein administration of the PD-1 antagonistand DR5 agonist reduces tumor size by at least 50%.
 43. A method oftreating cancer in a subject, the method comprising administering to thesubject an effective amount of a PD-1 antagonist and a DR5 agonist,wherein: (a) the PD-1 antagonist is an antibody comprising the CDR1,CDR2 and CDR3 domains in a heavy chain variable region having thesequence set forth in SEQ ID NO: 13, and the CDR1, CDR2 and CDR3 domainsin a light chain variable region having the sequence set forth in SEQ IDNO: 15 or an antibody comprising the CDR1, CDR2 and CDR3 domains in aheavy chain variable region having the sequence set forth in SEQ ID NO:1, and the CDR1, CDR2 and CDR3 domains in a light chain variable regionhaving the sequence set forth in SEQ ID NO: 3; and (b) the DR5 agonistis an antibody.
 44. The method of claim 31, wherein the PD-1 antagonistand the DR5 agonist are administered separately.
 45. The method of claim31, wherein the DR5 agonist and the PD-1 antagonist are administeredsimultaneously.
 46. The method of claim 31, wherein the cancer is acancer selected from the group consisting of leukemia, lymphoma,blastoma, carcinoma and sarcoma.
 47. The method of claim 31, whichcomprises administration of an additional therapeutic agent.
 48. Acomposition comprising a PD-1 antagonist and a DR5 agonist.
 49. A methodof treating cancer in a subject, comprising administering the subjectthe composition of claim
 48. 50. A kit for treating a cancer in asubject, the kit comprising: (a) a dose of a PD-1 antagonist; (b) a doseof a DR5 agonist; and (c) instructions for using the PD-1 antagonist andDR5 agonist in the method of claim 31.